Methods and compositions for simultaneous saccharification and fermentation

ABSTRACT

The invention provides compositions and methods for the synergistic degradation of oligosaccharides by endoglucanases. The invention further provides recombinant host cells containing one or more genes encoding endoglucanses which are capable of the synergistic degradation of oligosaccharides. Preferred host cells of the invention are ethanologenic and capable of carrying out simultaneous saccharification and fermentation resulting in the production of ethanol from complex cellulose substrates

RELATED INFORMATION

This application is a divisional application of U.S. application Ser.No. 09/885,297, filed Jun. 19, 2001, issuing, which claims priority toU.S. provisional application No. 60/214,137, entitled “SynergisticHydrolysis of Carboxymethyl Cellulose and Acid Swollen Cellulose by TwoEndoglucanases (EGZ and EGY),” filed Jun. 26, 2000, and U.S. provisionalapplication No. 60/219,913, entitled “Methods and Compositions forSimultaneous Saccharification and Fermentation,” filed Jul. 21, 2000,all of which are incorporated herein in their entirety by thisreference. The contents of all patents, patent applications, andreferences cited throughout this specification are hereby incorporatedby reference in their entireties.

GOVERNMENT SPONSORED RESEARCH

This work was supported, in part, by grants from the U.S. Department ofAgriculture, National Research Initiative (98-35504-6177 and98-35505-6976), the U.S. Department of Energy, Office of Basic EnergyScience (FG02-96ER20222), and the Florida Agricultural ExperimentStation, University of Florida.

BACKGROUND OF THE INVENTION

Many environmental and societal benefits would result from thereplacement of petroleum-based automotive fuels with renewable fuelsobtained from plant materials (Lynd et al., (1991) Science251:1318-1323; Olson et al., (1996) Enzyme Microb. Technol. 18:1-17;Wyman et al., (1995) Amer. Chem. Soc. Symp. 618:272-290). Each year, theUnited States burns over 120 billion gallons of automotive fuel, roughlyequivalent to the total amount of imported petroleum. The development ofethanol as a renewable alternative fuel has the potential to eliminateUnited States dependence on imported oil, improve the environment, andprovide new employment (Sheehan, (1994) ACS Symposium Series No. 566,ACS Press, pp 1-53).

In theory, the solution to the problem of imported oil for automotivefuel appears quite simple. Rather than using petroleum, a finiteresource, ethanol, a renewable resource, can be produced efficiently bythe fermentation of plant material. Indeed, Brazil has demonstrated thefeasibility of producing ethanol and the use of ethanol as a primaryautomotive fuel for more than 20 years. Similarly, the United Statesproduces over 1.2 billion gallons of fuel ethanol each year. Currently,fuel ethanol is produced from corn starch or cane syrup utilizing eitherSaccharomyces cerevisiae or Zymomonas mobilis (Z. mobilis). However,neither of these sugar sources can supply the volumes needed to realizea replacement of petroleum-based automotive fuels. In addition, bothcane sugar and corn starch are relatively expensive starting materials,which have competing uses as food products.

Moreover, these sugar substrates represent only a fraction of the totalcarbohydrates in plants. Indeed, the majority of the carbohydrates inplants are in the form of lignocellulose, a complex structural polymercontaining cellulose, hemicellulose, pectin, and lignin. Lignocelluloseis found in, for example, the stems, leaves, hulls, husks, and cobs ofplants. Hydrolysis of these polymers releases a mixture of neutralsugars including glucose, xylose, mannose, galactose, and arabinose. Noknown natural organism can rapidly and efficiently metabolize all ofthese sugars into ethanol.

Nonetheless, in an effort to exploit this substrate source, the Gulf OilCompany developed a method for the production of ethanol from celluloseusing a yeast-based process termed simultaneous saccharification andfermentation (SSF) (Gauss et al. (1976) U.S. Pat. No. 3,990,944). Fungalcellulase preparations and yeasts were added to a slurry of thecellulosic substrate in a single vessel. Ethanol was producedconcurrently during cellulose hydrolysis. However, Gulf's SSF processhas some shortcomings. For example, fungal cellulases have beenconsidered, thus far, to be too expensive for use in large scalebioethanol processes (Himmel et al., (1997) Amer. Chem. Soc. pp. 2-45;Ingram et al., (1987) Appl. Environ. Microbiol. 53:2420-2425; Okamoto etal., (1994) Appl. Microbiol. Biotechnol. 42:563-568; Philippidis, G.,(1994) Amer. Chem. Soc. pp. 188-217; Saito et al., (1990) J. Ferment.Bioeng. 69:282-286; Sheehan, J., (1994) Amer. Chem. Soc. pp 1-52; Su etal., (1993) Biotechnol. Lett. 15:979-984).

SUMMARY OF THE INVENTION

The development of inexpensive enzymatic methods for cellulosehydrolysis has great potential for improving the efficiency of substrateutilization and the economics of the saccharification and fermentationprocess. Accordingly, developing enzymes and, preferably, biocatalyststhat produce such enzymes which can be used for the efficientdepolymerization of a complex sugars and subsequent rapid fermentationof the sugar into alcohol, would be of great benefit.

Certain microbes, such as Erwinia chrysanthemi, produce a number ofhydrolase and lyase enzymes, which are very effective in the degradingof plant tissues containing complex sugars. In particular, this organismproduces two different endoglucanase activities (comprising EGY and EGZ)which have been discovered to function, when used in particular amounts,as highly effective enzyme compositions for degrading complex sugars.These enzymes may be used as crude extracts having a desired mixture ofendoglucanase activity or, preferably, may be used as purifiedcompositions.

Moreover, a biocatalyst, preferably a recombinant bacterium, morepreferably a ethanologenic bacterium, can be engineered to express oneor more of these enzymatic activities in particular amounts sufficientfor degrading complex sugars. Such a biocatalyst is suitable for theefficient degradation of complex sugars and subsequent fermentation intoalcohol by a process known as simultaneous saccharification andfermentation (SSF). An advantage of the above endoglucanase compositionsor biocatalysts is that the need for additional fungal cellulases fordegrading the complex sugars is reduced or eliminated.

The present invention provides endoglucanase activities for carrying outthe degrading of a complex sugar and more preferably, the use ofendoglucanase activities in particular ratios for optimal degrading of acomplex sugar.

In addition, the invention provides recombinant host cells engineeredfor optimal expression and secretion of endoglucanase activitiessuitable for degrading complex sugars. Specifically exemplified arerecombinant enteric bacteria, Escherichia and Klebsiella, which expressan endoglucanase under the transcriptional control of a surrogatepromoter for optimal expression. In addition, also exemplified is arecombinant enteric bacterium that expresses two differentendoglucanases celY and celZ, where each is under the transcriptionalcontrol of a surrogate promoter for optimal expression in a particularratio.

The invention provides for the further modification of these hosts toinclude a secretory protein/s that allow for the increased productionand/or secretion of the endoglucanases from the cell. In a preferredembodiment, the invention provides for the further modification of thesehosts to include exogenous ethanologenic genes derived from an efficientethanol producer, such as Zymomonas mobilis.

Accordingly, these hosts are capable of expressing high levels ofproteins that may be used alone or in combination with other enzymes orrecombinant hosts for the efficient production of alcohol from complexsugars.

More particularly, in a first aspect, the invention provides acomposition for degrading an oligosaccharide containing, a firstendoglucanase having a first degrading activity, and a secondendoglucanase having a second degrading activity, where the first andsecond degrading activities are present in a ratio such that thedegrading of the oligosaccharide by the first and second endoglucanasesis synergized.

In a second aspect, the invention provides a method for degrading anoligosaccharide comprising, contacting an oligosaccharide with a firstendoglucanase having a first degrading activity and a secondendoglucanase having a second degrading activity, where the first andsecond degrading activities are present in a ratio such that thedegrading of the oligosaccharide by the first and second endoglucanasesis synergized.

In one embodiment of the above aspects, the contacting of theoligosaccharide with the first endoglucanase and the secondendoglucanase is performed in any order or concurrently.

In one embodiment of the above aspects, the first endoglucanase or thesecond endoglucanase, or both the first and the second endoglucanases,are derived from a cell extract. The cell extract is derived from abacterial cell, e.g., a bacterial cell that has been recombinantlyengineered to express the first endoglucanase or the secondendoglucanase, or both the first and the second endoglucanases. In arelated embodiment, the bacterial cell is selected from the familyEnterobacteriaceae, and preferably, is either Escherichia or Klebsiella,and more preferably contains a first endoglucanase that is encoded bycelZ and a second endoglucanase that is encoded by celY, and where celZand celY are derived from Erwinia.

In another embodiment of the above aspects, the first endoglucanase isEGZ and the second endoglucanase is EGY, preferably in a ratio rangingfrom about 1:1 to, more preferably, about 9:1 to about 19:1.

In still another embodiment of the above aspects, the firstendoglucanase or the second endoglucanase, or both the first and thesecond endoglucanase, are purified.

In even another embodiment of the above aspects, the degrading of anoligosaccharide is synergized by a factor ranging from about 1.1 toabout 2.0, and preferably by about 1.8.

In yet another embodiment of the above aspects, the composition containsan additional enzyme, e.g., an endoglucanase, exoglucanase,cellobiohydrolase, β-glucosidase, endo-1,4-β-xylanase, α-xylosidase,α-glucuronidase, α-L-arabinofuranosidase, acetylesterase,acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase,β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase,pectate lyase, or a combination thereof.

In a related embodiment of the above aspects, the additional enzyme is aglucanase derived from a fungus, preferably T. longibranchiatum.

In another related embodiment of the above aspects, the additionalenzyme is an ethanologenic enzyme, preferably an ethanologenic enzymesuch as pyruvate decarboxylase or alcohol dehydrogenase.

In another embodiment of the above aspects, the first endoglucanase andthe second endoglucanase are packaged separately.

In another embodiment of the above aspects, the composition is used forsimultaneous saccharification and fermentation.

In still another embodiment of the above aspects, the oligosaccharide isa cellooligosaccharide, lignocellulose, hemicellulose, cellulose,pectin, or any combination thereof.

In still another embodiment of the above aspects, the composition ormethod is, respectively, used or conducted in an aqueous solution.

In a third aspect, the invention provides a recombinant host cellsuitable for degrading an oligosaccharide containing a firstheterologous polynucleotide segment encoding a first endoglucanasehaving a first degrading activity, where the segment is under thetranscriptional control of a surrogate promoter; and a secondheterologous polynucleotide segment encoding a second endoglucanasehaving a second degrading activity, where the segment is under thetranscriptional control of a surrogate promoter, and where the firstendoglucanase and the second endoglucanase are expressed so that thefirst and the second degrading activities are present in a ratio suchthat the degrading of the oligosaccharide by the first and secondendoglucanases is synergized.

In one embodiment, the first endoglucanase or the second endoglucanase,or both the first and the second endoglucanases are secreted. In arelated embodiment, the first endoglucanase or the second endoglucanase,or both the first and the second endoglucanases, are derived from a cellextract. The cell extract is derived from a bacterial cell, e.g., abacterial cell that has been recombinantly engineered to express thefirst endoglucanase or the second endoglucanase, or both the first andthe second endoglucanases. In a related embodiment, the bacterial cellis selected from the family Enterobacteriaceae, and preferably, iseither Escherichia or Klebsiella, and more preferably E. coli B, E. coliDH5α, or Klebsiella oxytoca.

In yet another embodiment of the above aspects, the composition containsan additional enzyme, e.g., an endoglucanase, exoglucanase,cellobiohydrolase, β-glucosidase, endo-1,4-β-xylanase, α-xylosidase,α-glucuronidase, α-L-arabinofuranosidase, acetylesterase,acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase,β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase,pectate lyase, or a combination thereof.

In one embodiment, the first endoglucanase is encoded by celZ and thesecond endoglucanase is encoded by celY, and where celZ and celY arederived from Erwinia.

In a related embodiment of the above aspects, the additional enzyme is aglucanase derived from a fungus, preferably T. longibranchiatum.

In another related embodiment, the first endoglucanase is EGZ and thesecond endoglucanase is EGY.

In another related embodiment, the additional enzyme is a secretoryenzyme, preferably a pul or out gene product.

In another related embodiment of the above aspects, the host cell isethanologenic, e.g., E. coli KO4 (ATCC 55123), E. coli KO 11 (ATCC55124), E. coli KO12 (ATCC 55125) and E. coli LY01 (ATCC 11303), K.oxytoca M5A1, and K. oxytoca P2 (ATCC 55307).

In a fourth aspect, the invention provides a method for enhancing thedegradation of an oligosaccharide by contacting an oligosaccharide witha host cell containing, a first heterologous polynucleotide segmentencoding a first endoglucanase having a first degrading activity, wherethe segment is under the transcriptional control of a surrogatepromoter; and a second heterologous polynucleotide segment containing asequence encoding a second endoglucanase having a second degradingactivity, where the segment is under the transcriptional control of asurrogate promoter. The method further provides that the firstendoglucanase and the second endoglucanase are expressed so that thefirst and the second degrading activities are present in a ratio suchthat the degrading of the oligosaccharide by the first and secondendoglucanases is synergized and thereby enhanced.

In one embodiment of the above aspect, the first endoglucanase or thesecond endoglucanase or both the first and the second endoglucanases aresecreted.

In another embodiment, the host cell of the above method isethanologenic.

In another embodiment, the method is conducted in an aqueous solution.

In even another embodiment, the method is used for simultaneoussaccharification and fermentation.

In yet another embodiment, the method includes degrading anoligosaccharide selected from the group consisting ofcellooligosaccharide, lignocellulose, hemicellulose, cellulose, pectin,or any combination thereof.

In a fifth aspect, the invention provides a method of making arecombinant host cell suitable for degrading an oligosaccharide byintroducing into the host cell a first heterologous polynucleotidesegment encoding a first endoglucanase having a first degradingactivity, where the segment is under the transcriptional control of asurrogate promoter; and a second heterologous polynucleotide segmentcontaining a sequence encoding a second endoglucanase having a seconddegrading activity, where the segment is under the transcriptionalcontrol of a surrogate promoter. The method further provides that thefirst and second endoglucanases are expressed such that the first andthe second degrading activities are present in a ratio such that thedegrading of the oligosaccharide by the first and second endoglucanasesis synergized.

In one embodiment of the above aspect, the first endoglucanase or thesecond endoglucanase or both the first and second endoglucanases aresecreted.

In another embodiment, the host cell is ethanologenic.

In even another embodiment, the first endoglucanase is encoded by celZand the second endoglucanase is encoded by celY, and celZ and celY arederived from Erwinia.

In still another embodiment, the surrogate promoter of the firstheterologous polynucleotide segment or the second heterologouspolynucleotide segment or both the first and second polynucleotidesegments, contains a polynucleotide fragment derived from Zymomonasmobilis.

In yet another embodiment, the recombinant host cell is suitable forsimultaneous saccharification and fermentation, and preferably, isethanologenic.

In a sixth aspect, the invention provides a method for making arecombinant host cell integrant by introducing into the host cell avector containing the polynucleotide sequence of pLOI2352 (SEQ ID NO:17) and identifying a host cell having the vector stably integrated.

In a seventh aspect, the invention provides a method for expressing aendoglucanase in a host cell by introducing into the host cell a vectorcontaining the polynucleotide sequence of pLOI2306 (SEQ ID NO: 12) andidentifying a host cell expressing the endoglucanase.

In an eighth aspect, the invention provides a method for producingethanol from an oligosaccharide source by contacting the oligosaccharidesource with a ethanologenic host cell containing a first heterologouspolynucleotide segment encoding a first endoglucanase having a firstdegrading activity, where the segment is under the transcriptionalcontrol of a surrogate promoter; and a second heterologouspolynucleotide segment encoding a second endoglucanase having a seconddegrading activity, where the segment is under the transcriptionalcontrol of a surrogate promoter. The method further provides that thefirst and second endoglucanases are expressed so that the first and thesecond degrading activities are present in a ratio such that thedegrading of the oligosaccharide by the first and second endoglucanasesis synergized resulting in a degraded oligosaccharide that is fermentedinto ethanol.

In one embodiment, the first endoglucanase is encoded by celZ and thesecond endoglucanase is encoded by celY gene, and celZ and celY arederived from Erwinia.

In another embodiment, the host cell further contains a heterologouspolynucleotide segment encoding at least one pul gene or out gene.

In even another embodiment, the host cell is selected from the familyEnterobacteriaceae, preferably Escherichia or Klebsiella, morepreferably E. coli KO4 (ATCC 55123), E. coli KO11 (ATCC 55124), E. coliKO12 (ATCC 55125), LY01 (ATCC 11303), K. oxytoca M5A1, or K. oxytoca P2(ATCC 55307).

In another embodiment, the method is conducted in an aqueous solution.

In still another embodiment, the oligosaccharide is selected from thegroup consisting of cellooligosaccharide, lignocellulose, hemicellulose,cellulose, pectin, and any combination thereof.

In another embodiment, the heterologous polynucleotide segment is, orderived from, pLOI 2352 (SEQ ID NO: 17).

In yet another embodiment, the first endoglucanase is EGZ and the secondendoglucanase is EGY.

In another embodiment, the surrogate promoter of the firstpolynucleotide segment or the second polynucleotide segment, or both thefirst and the second polynucleotide segments contains a polynucleotidefragment derived from Zymomonas mobilis.

In ninth aspect, the invention provides a vector containing thepolynucleotide sequence of a plasmid, or fragment thereof, of pLOI23 11,pLOI1620, pLOI2316, pLOI2317, pLOI2318, pLOI2319, pLOI2320, pLOI2323,pLOI2342, pLOI2348, pLOI2349, pLOI2350, pLOI2352, pLOI2353, pLOI2354,pLOI2355, pLOI2356, pLOI2357, pLOI2358, or pLOI2359.

In a tenth aspect, the invention provides a host cell containing avector having the polynucleotide sequence of a plasmid, of fragmentthereof, of pLOI2311, pLOI1620, pLOI2316, pLOI2317, pLOI2318, pLOI2319,pLOI2320, pLOI2323, pLOI2342, pLOI2348, pLOI2349, pLOI2350, pLOI2352,pLOI2353, pLOI2354, pLOI2355, pLOI2356, pLOI2357, pLOI2358, or pLOI2359.

In one embodiment, the host is Klebsiella oxytoca strain P2 (pCPP2006),Klebsiella oxytoca strain SZ6 (pCPP2006), Klebsiella oxytoca strain SZ21(pCPP2006), or Klebsiella oxytoca strain SZ22 (pCPP2006).

In an eleventh aspect, the invention provides a method for degrading anoligosaccharide by obtaining a first endoglucanase having a firstdegrading activity, obtaining a second endoglucanase having a seconddegrading activity, and contacting an oligosaccharide with the first andsecond endoglucanases, where the first and second degrading activitiesare present in a ratio such that the degrading of the oligosaccharide bythe first and second endoglucanases is synergized.

In a twelfth aspect, the invention provides a method for enhancing thedegrading of an oligosaccharide by contacting an oligosaccharide with afirst endoglucanase having a first degrading activity and a secondendoglucanase having a second degrading activity, where the first andsecond degrading activities are present in a ratio such that thedegrading of the oligosaccharide by the first and second endoglucanasesis synergized and thereby enhanced.

In a related aspect, the invention provides a method for degrading,and/or for enhancing the degrading of, an oligosaccharide by contactingan oligosaccharide with a first endoglucanase having a first degradingactivity and a second endoglucanase having a second degrading activity,where the first and second degrading activities are present in a ratiosuch that the degrading of the oligosaccharide by the first and secondendoglucanases results in a change in viscosity, preferably, a reductionin viscosity, more preferably by an amount of at least, e.g., 5centopoise, 10 centopoise, 20 centopoise, 50 centopoise, 100 centopoise,500 centopoise, or 1000 centopoise or more, or within a range thereof.

In a preferred embodiment, the oligosaccharide is cellulose, e.g.,amorphous cellulose or crystalline cellulose and may be from a sourcesuch as, e.g., paper, pulp, or plant fiber.

In a thirteenth aspect, the invention provides a recombinant host cellsuitable for degrading an oligosaccharide containing a firstheterologous polynucleotide segment encoding a first endoglucanase; anda second heterologous polynucleotide segment encoding a secondendoglucanase.

In a related aspect, the host cell is suitable for reducing theviscosity of an oligosaccharide by comprising a first heterologouspolynucleotide segment encoding a first endoglucanase; and a secondheterologous polynucleotide segment encoding a second endoglucanase.

In one embodiment, the first heterologous polynucleotide segment isunder the transcriptional control of a surrogate promoter, and thesecond heterologous polynucleotide segment is under the transcriptionalcontrol of a surrogate promoter.

In another embodiment, the cell is a bacterial cell, preferably selectedfrom the family Enterobacteriaceae, more preferably from the genusEscherichia or Klebsiella.

In another embodiment, the first endoglucanase is encoded by celZ andthe second endoglucanase is encoded by celY, and celZ and celY arederived from Erwinia.

In another embodiment, the first endoglucanase is EGZ and said secondendoglucanase is EGY.

In a fourteenth aspect, the invention provides a recombinant host strainof Klebsiella oxytoca strain P2 (pCPP2006) represented by a deposit withthe American Type Culture Collection designated as deposit number ATCCPTA-3468.

In a fifteenth aspect, the invention provides a recombinant host strainof Klebsiella oxytoca strain SZ6 (pCPP2006) represented by a depositwith the American Type Culture Collection designated as deposit numberATCC PTA-3464.

In a sixteenth aspect, the invention provides a recombinant host strainof Klebsiella oxytoca strain SZ21 (pCPP2006) represented by a depositwith the American Type Culture Collection designated as deposit numberATCC PTA-3465.

In a seventeenth aspect, the invention provides a recombinant hoststrain of Klebsiella oxytoca strain SZ22 (pCPP2006) represented by adeposit with the American Type Culture Collection designated as depositnumber ATCC PTA3467.

In a eighteenth aspect, the invention provides a recombinant cellcontaining a first heterologous polynucleotide segment encoding a firstendoglucanase; and a second heterologous polynucleotide segment encodinga second endoglucanase, where the first polynucleotide segment encodinga first endoglucanase or the second polynucleotide segment encoding asecond endoglucanase, or both are sufficiently homologous in an aminoacid alignment to either the gene product of celY or celZ from Erwiniaas to share the functional activity of being capable of degrading apolysaccharide.

In nineteenth aspect, the invention provides an extract or compositionderived from a host cell of the invention, e.g., a secreted polypeptide,a lysate or broth, or a pure, semi-pure, or unpurified enzymatic extractor polypeptide which is suitable for degrading and/or reducing theviscosity of a oligosaccharide when contacted thereto.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fermentation rates for the ethanologenic recombinant hostE. coli KO11 using rice hull substrates pretreated with dilute acid andsupplemented with two different media.

FIG. 2 shows simultaneous saccharification and fermentation (SSF) ratesfor the ethanologenic recombinant host strain K. oxytoca P2 using mixedwaste office paper. Insoluble residues from SSF were recycled as asource of bound cellulase enzymes and substrate during subsequentfermentations.

FIG. 3 shows the structure of the plasmid pLOI2171, a low copy promoterprobe vector showing the orientation of the kanamycin resistance gene(kan) for selection, the temperature sensitive pSC101 replicon (Rep(ts))for episomal maintenance of the plasmid, and the promoterlesspolysaccharase gene celZ encoding phospho-beta-glucosidase (EGZ).

FIG. 4 is a graph showing the high correspondence between the size ofthe zone of clearance on CMC indicator plates (x-axis) measured for atransformed bacterial colony and the amount of glucanase activityexpressed (y-axis).

FIG. 5 shows the partial nucleotide sequence (SEQ ID NO: 1) of the Z.mobilis DNA fragment in the pLOI2183 plasmid that functions as asurrogate promoter. The full sequence has been assigned GenBankAccession Number AF109242 (SEQ ID NO: 2). Indicated are twotranscriptional start sites (#), −35 and −10 regions, the Shine-Delgarnosite (bold), partial vector and celZ sequence (lowercase), and the celZstart codon (atg indicated in bold).

FIG. 6 represents electron micrographs of E. coli DH5α cells harboringdifferent plasmids expressing little if any (pUC19; panel A), moderate(pLOI2164; panel B), and high levels (pLOI2307; panel C) of glucanase inthe form of periplasmic inclusion bodies (pib) localized between theouter cell wall and the inner membrane (im). The bar shown represents0.1 μm.

FIG. 7 shows a schematic detailing the cloning strategy used toconstruct the celZ integration vector pLOI2306, a genetic constructcapable of being introduced into the genome of a recombinant host andconferring stable glucanase expression activity to the host.

FIG. 8 shows a schematic representation of the celZ integration vectorpLOI2306 (SEQ ID NO: 12) with the locations of the surrogate promoterfrom Z. mobilis, the celZ gene from E. chrysanthemi, resistance markers(bla and tet), and K. oxytoca target sequence indicated.

FIG. 9 shows graphical depictions of the synergistic action of EGY andEGZ. Both enzymes were diluted to equal CMCase activities (1.5 IU/ml)with calculated synergies shown in parenthesis. Panel A shows a graphdepicting the effect of enzyme ratios on synergy. Different amounts ofEGY and EGZ were combined to maintain a constant predicted activity(0.15 IU/ml) based on the contribution of individual enzymes. Assayswere incubated with CMC for 1 hour at 35° C. and terminated by boiling.Numbers on the X axis indicate the proportions of EGZ and EGY. Synergyis shown above each bar. Panel B shows a graph depicting the hydrolysisof CMC by EGZ and EGY, alone and in combination (9 parts EGZ+1 partEGY). All assays contained equal total activities (0.15 IU/ml) based onthe sum of individual EGY and EGZ activities. Synergy is shown aboveeach point for the combination of both enzymes. Panel C shows a graphdepicting the hydrolysis of acid-swollen cellulose by EGZ and EGY, aloneand in combination. A 9 to 1 ratio of EGZ to EGY was used for thecombined enzyme reaction. All assays contained 1.5 IU/ml based on thesum of individual EGY and EGZ activities. Synergy is shown above eachpoint for the combination of both enzymes.

FIG. 10 represents a thin layer chromatography (TLC) analysis of thehydrolysis products from two complex sugars: acid-swollen cellulose andAvicel®. Approximately 1.5 IU and 25 IU of CMCase were used in reactionswith acid-swollen cellulose and Avicel®, respectively. Abbreviations forY axis: G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose;and G5, cellopentaose. Lanes: S, mixed cellooligosaccharide standard; C,control lacking enzyme; Z, EGZ; Y, EGY, and Z+Y: EGZ+EGY. Panel A showsresults with acid-swollen cellulose after a 6-hour incubation withCMCase (1 μl loading). Panel B shows results with acid-swollen celluloseafter a 6-hour incubation with CMCase (2 μl loading). Panel C showsresults with Avicel® after a 48-hour incubation with CMCase (10 μlloading).

FIG. 11 represents a TLC analysis showing the hydrolysis ofcellooligosaccharides by EGZ and EGY. Each test contained approximately0.07 IU of CMCase per ml (2 hour incubation, 35° C.). Abbreviations: S,mixed cellooligosaccharides standard; G1, glucose; G2, cellobiose; G3,cellotriose; G4, cellotetraose; and G5, cellopentaose. Panel A shows thesubstrates before hydrolysis, Panel B shows the substrates afterincubation with EGY, Panel C shows the substrates after incubation withEGZ, and Panel D shows EGZ hydrolysis of cellopentaose after differentperiods of incubation (0, 5, 10, and 25).

FIG. 12 is a model illustrating the utilization of amorphous celluloseby E. chrysanthemi. Three glucosidases are used for the catabolism ofamorphous cellulose. Two of these, EGY and EGZ are extracellularendoglucanases, which function, together in a synergistic fashion. EGYrequires large substrate molecules and hydrolyzes these into shorter,insoluble fragments. EGY does not hydrolyze solublecellooligosaccharides (2 to 5 glucosyl residues). EGZ readily hydrolyzessoluble cellooligosaccharides (cellopentaose and cellotetraose) andamorphous fragments of intermediate length to produce cellobiose andcellotriose. Cellobiose and cellotriose are phosphorylated duringcellular uptake by a phosphoenolpyruvate-dependent phosphotransferasesystem. Hydrolysis is completed intracellularly by a third enzyme,phospho-β-glucosidase. Resulting monomeric products (glucose andglucose-6-phosphate) are metabolized by glycolysis.

FIG. 13 is a schematic representation of the construction of apromoter-probe vector for celY. Sau3AI fragments of Z. mobilischromosomal DNA were ligated into the BamHI site of pLOI2317 to providea strong, surrogate promoter for celY coding region (solid segments). Z.mobilis DNA fragments (promoter 1 and promoter 2) are shown as opensegments. Replicons and antibiotic resistance genes are stippled; othervector DNA is shown as thin connecting lines. Arrows indicate directionof transcription.

FIG. 14 is a depiction of transcriptional initiation sites and putativepromoter regions for the celY promoter in DH5α (pLOI2323).Transcriptional starts for celY were identified by primer extensionanalysis. Four promoters (SEQ ID NOS 18-21, respectively in order ofappearance) were identified. Upstream sequences of these promoters withsimilarity to E. coli −35 and −10 regions are marked with underlines.RNA start sites are bolded. Putative promoters are numbered inparenthesis adjacent to the start site in descending order from thestrongest. Differences in intensities were small, with 2-fold.

FIG. 15 is a schematic representation of the construction of pLOI2352for the functional integration of celY and celZ into the chromosome ofethanologenic K. oxytoca P2. Coding regions for celY and celZ are shownas solid segments. Fragments of Z. mobilis DNA that serve as promoters(prom1 and prom2) are shown as open segments. Replicons and antibioticresistance genes are stippled; other vector DNA is shown as a thinconnecting line. Arrows on segments indicate the direction oftranscription. The small open arrows represent the FRT sites, which arerecognized by the flp recombinase. FRT sequences are asymmetrical andarranged to allow the deletion of plasmid DNA (replicon and selectablemarker) after chromosomal integration.

FIG. 16 is a schematic representation of the construction of pLO12357for the inactivation of celZ by double homologous recombination. Codingregions for celY are shown as solid segments. The fragment of Z. mobilisDNA that serves as a promoter (prom1) is shown as open segments.Replicons and antibiotic resistance genes are stippled; other vector DNAis shown as a thin connecting line. Arrows on segments indicate thedirection of transcription. The small open arrows represent the FRTsites, which are recognized by the flp recombinase.

FIG. 17 shows a digital image of a thin layer chromatogram offermentation broth illustrating the utilization of cellobiosides. Theleft panel (A) represents strain P2(pCPP2006), the parent lacking E.chrysanthemi endoglucanases, whereas the right panel (B) representsstrain SZ21(pCPP2006), the recombinant secreting high levels of CelY andCelZ endoglucanases. Approximately 4 μL of broth was spotted in eachlane and labels G1 through G6 refer to the number of glucosyl residuesin the adjacent spots. Lanes for both panels (A and B) are from left toright: I, initial (0 h); 2, after 10 h; and 3, after 36 h offermentation.

FIG. 18 shows a graphical depiction of the amount of ethanol productionfrom amorphous cellulose by ethanologenic derivatives of K. oxytocaM5A1. Results shown in the top and middle panel (A and B) includestandard deviations from three replicates; results shown in the bottompanel (C) represent single fermentations. The top panel (A) showsresults from the fermentation of 6.85 g/L amorphous cellulose; themiddle panel (B) shows results from the fermentation of 15.33 g/Lamorphous cellulose; and the bottom panel (C) shows the fermentation of28.96 g/L amorphous cellulose. Strain P2(pCPP2006) is the parentalstrain and lacks E. chrysanthemi endoglucanase genes. StrainSZ6(pCPP2006) secretes CelZ endoglucanase; strain SZ21 (pCPP2006)secretes CelY and CelZ endoglucanases; strain SZ22(pCPP2006) secretesCelY endoglucanase.

DETAILED DESCRIPTION OF THE INVENTION

In order for the full scope of the invention to be clearly understood,the following definitions are provided.

I. Definitions

As used herein the term “recombinant host” is intended to include a cellsuitable for genetic manipulation, e.g., which can incorporateheterologous polynucleotide sequences, e.g., which can be transfected.The cell can be a microorganism or a higher eukaryotic cell. The term isintended to include progeny of the cell originally transfected. Inpreferred embodiments, the cell is a bacterial cell, e.g., aGram-negative bacterial cell, and this term is intended to include allfacultatively anaerobic Gram-negative cells of the familyEnterobacteriaceae such as Escherichia, Shigella, Citrobacter,Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia,Cedecea, Morganella, Hafnia, Edwardsiella, Providencia, Proteus, andYersinia. Particularly preferred recombinant hosts are Escherichia colior Klebsiella oxytoca cells.

The term “ratio” is intended to include the relationship between theamounts (measured, e.g., by activity or in moles) of two enzymes in apredetermined combination where, preferably, the ratio is not naturallyoccurring and, more preferably, results in synergistic enzyme activity.

The terms “a first endoglucanase having a first degrading activity” and“a second endoglucanase having a second degrading activity” are intendedto include, respectively, an endoglucanase with an activity that can bedistinguished from another endoglucanase (e.g., a second endoglucanase)with a second activity functionally (e.g., by its activity on aparticular substrate; synergism with another enzyme), by source oforigin (e.g., host cell strain, including naturally occurring strains orgenetically modified strains expressing a clone expressing anendoglucanase), or by biochemical properties using art recognizedtechniques (e.g., molecular weight determination or purificationcharacteristics). The degrading activity, e.g., the enzymatic hydrolysisof an oligosaccharide, can also comprise a change in the viscosity ofthe oligosaccharide.

The terms “synergism,” “synergistic activity,” and “synergized” areintended to describe the interaction between distinguishablepolypeptides or polypeptide activities wherein the effect of the totalactivity of the polypeptides taken together are greater than the sum ofthe effects of the individual activities. The polypeptides may be, forexample, endoglucanases, exoglucanases, cellobiohydrolases,β-glucosidases, endo-1,4-β-xylanases, α-xylosidases, α-glucuronidases,α-L-arabinofuranosidases, acetylesterases, acetylxylanesterases,α-amylases, β-amylases, glucoamylases, pullulanases, β-glucanases,hemicellulases, arabinosidases, mannanases, pectin hydrolases, pectatelyases, or any combination thereof. An activity of a polypeptideincludes the degradation (e.g., hydrolysis) of an oligosaccharide butmay also include a change in the viscosity of the oligosaccharide. Thesynergized degrading of an oligosaccharide is preferably by a factor ofabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or more preferablyabout 2.0, with a typical factor being about 1.8.

The term “heterologous polynucleotide segment” is intended to include apolynucleotide segment that encodes one or more polypeptides or portionsor fragments of polypeptides. A heterologous polynucleotide segment maybe derived from any source, e.g., eukaryotes, prokaryotes, virii, orsynthetic polynucleotide fragments.

The terms “polysaccharase,” “cellulase,” or “glucanase” are usedinterchangeably herein and are intended to include a polypeptide capableof catalyzing the degradation or depolymerization of any linked sugarmoiety, e.g., disaccharides, trisaccharides, oligosaccharides,including, complex carbohydrates, also referred to herein as complexsugars, e.g., cellooligosaccharide and lignocellulose, which comprisescellulose, hemicellulose, and pectin. The terms are intended to includecellulases such as glucanases, including preferably, endoglucanases butalso including, e.g., exoglucanase, β-glucosidase, cellobiohydrolase,endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase,α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase,β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase,arabinosidase, mannanase, pectin hydrolase, pectate lyase, or acombination of any of these cellulases.

The term “endoglucanase” is intended to include a cellulase whichtypically hydrolyses internal β1-4 glucosyl linkages in polymericsubstrates and does not preferentially hydrolyze linkages located at theends of the chain.

The term “surrogate promoter” is intended to include a polynucleotidesegment that can transcriptionally control a gene-of-interest that itdoes not transcriptionally control in nature. In a preferred embodiment,the transcriptional control of a surrogate promoter results in anincrease in expression of the gene-of-interest. In a preferredembodiment, a surrogate promoter is placed 5′ to the gene-of-interest. Asurrogate promoter may be used to replace the natural promoter, or maybe used in addition to the natural promoter. A surrogate promoter may beendogenous with regard to the host cell in which it is used or it may bea heterologous polynucleotide sequence introduced into the host cell,e.g., exogenous with regard to the host cell in which it is used. Otherpromoters suitable for use in bacteria include, e.g., lacZ, T7, and SP6(see, e.g., Ausubel et al. infra).

The terms “oligosaccharide source,” “oligosaccharide,” “complexcellulose,” “complex carbohydrate,” and “complex sugar,” and“polysaccharide” are used essentially interchangeably and are intendedto include any carbohydrate source comprising more than one sugarmolecule. These carbohydrates may be derived from any unprocessed plantmaterial or any processed plant material. Examples are wood, paper,pulp, plant derived fiber, or synthetic fiber comprising more than onelinked carbohydrate moiety, i.e., one sugar residue. One particularoligosaccharide source is lignocellulose, which represents approximately90% of the dry weight of most plant material and contains carbohydrates,e.g., cellulose, hemicellulose, pectin, and aromatic polymers, e.g.,lignin. Cellulose makes up 30%-50% of the dry weight of lignocelluloseand is a homopolymer of cellobiose (a dimer of glucose). Similarly,hemicellulose, makes up 20%-50% of the dry weight of lignocellulose andis a complex polymer containing a mixture of pentose (xylose, arabinose)and hexose (glucose, mannose, galactose) sugars which contain acetyl andglucuronyl side chains. Pectin makes up I%-20% of the dry weight oflignocellulose and is a methylated homopolymer of glucuronic acid. Otheroligosaccharide sources include carboxymethyl cellulose (CMC), amorphouscellulose (e.g., acid-swollen cellulose), and the cellooligosaccharidescellobiose, cellotriose, cellotetraose, and cellopentaose. Cellulose,e.g., amorphous cellulose may be derived from a paper or pulp source(including, e.g., fluid wastes thereof) or, e.g., agriculturalbyproducts, e.g., corn stalks, soybean solubles, or beet pulp. Any oneor a combination of the above carbohydrate polymers are potentialsources of sugars for depolymerization and subsequent bioconversion toethanol by fermentation according to the products and methods of thepresent invention.

The term “gene/s” or “polynucleotide segment” is intended to includenucleic acid molecules, e.g., polynucleotides which include an openreading frame encoding a polypeptide, and can further include non-codingregulatory sequences, and introns. In addition, the terms are intendedto include one or more genes that map to a functional locus, e.g., theout or pul genes of Erwinia and Klebsiella, respectively, that encodemore than one gene product, e.g., a secretory polypeptide. In addition,the terms are intended to include a specific gene for a selectedpurpose. The gene may be endogenous to the host cell or may berecombinantly introduced into the host cell, e.g., as a plasmidmaintained episomally or a plasmid (or fragment thereof) that is stablyintegrated into the genome. In a preferred embodiment, the gene ofpolynucleotide segment is involved in at least one step in thebioconversion of a carbohydrate to ethanol. Accordingly, the term isintended to include any gene encoding a polypeptide such as an alcoholdehydrogenase, a pyruvate decarboxylase, a secretory protein/s, or apolysaccharase, e.g., a glucanase, such as an endoglucanase orexoglucanase, a cellobiohydrolase, β-glucosidase, endo-1,4-β-xylanase,β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase,acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase,β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase,pectate lyase, or a combination thereof.

The term “simultaneous saccharification and fermentation” or “SSF” isintended to include the use of one or more recombinant hosts (orextracts thereof, including purified or unpurified extracts) for thecontemporaneous degradation or depolymerization of a complex sugar andbioconversion of that sugar residue into ethanol by fermentation.

The term “transcriptional control” is intended to include the ability tomodulate gene expression at the level of transcription. In a preferredembodiment, transcription, and thus gene expression, is modulated byreplacing or adding a surrogate promoter near the 5′ end of the codingregion of a gene-of-interest thereby resulting in altered geneexpression. In a most preferred embodiment, the transcriptional controlof one or more gene is engineered to result in the optimal expression ofsuch genes, e.g., in a desired ratio. The term also includes inducibletranscriptional control as recognized in the art.

The term “expression” is intended to include the expression of a gene atleast at the level of mRNA production.

The term “expression product” is intended to include the resultantproduct of an expressed gene, e.g., a polypeptide.

The term “increased expression” is intended to include an alteration ingene expression at least at the level of increased mRNA production andpreferably, at the level of polypeptide expression.

The term “increased production” is intended to include an increase inthe amount of a polypeptide expressed, in the level of the enzymaticactivity of the polypeptide, or a combination thereof.

The terms “activity” and “enzymatic activity” are used interchangeablyand are intended to include any functional activity normally attributedto a selected polypeptide when produced under favorable conditions. Theactivity of an endoglucanase (e.g., EGY or EGZ) is, for example, theability of the polypeptide to enzymatically depolymerize a complexsaccharide. Typically, the activity of a selected polypeptideencompasses the total enzymatic activity associated with the producedpolypeptide. The polypeptide produced by a host cell and havingenzymatic activity may be located in the intracellular space of thecell, cell-associated, secreted into the extracellular milieu, or acombination thereof. Techniques for determining total activity ascompared to secreted activity are described herein and are known in theart.

The term “secreted” is intended to include an increase in the secretionof a polypeptide into the periplasmic space or into the extracellularmilieu, e.g., a heterologous polypeptide, preferably a polysaccharase.Typically, the polypeptide is secreted at an increased level that is inexcess of the naturally-occurring amount of secretion. More preferably,the term “secreted” refers to an increase in secretion of a givenpolypeptide that is at least 10% and more preferably, at least about100%, 200%, 300,%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more,as compared to the naturally-occurring level of secretion.

The term “secretory polypeptide” is intended to include anypolypeptide/s, alone or in combination with other polypeptides, thatfacilitate the transport of another polypeptide from the intracellularspace of a cell to the extracellular milieu. In one embodiment, thesecretory polypeptide/s encompass all the necessary secretorypolypeptides sufficient to impart secretory activity to a Gram-negativehost cell. Typically, secretory proteins are encoded in a single regionor locus that may be isolated from one host cell and transferred toanother host cell using genetic engineering. In a preferred embodiment,the secretory polypeptide/s are derived from any bacterial cell havingsecretory activity. In a more preferred embodiment, the secretorypolypeptide/s are derived from a host cell having Type II secretoryactivity. In another more preferred embodiment, the host cell isselected from the family Enterobacteriaceae. In a most preferredembodiment, the secretory polypeptide/s are one or more gene products ofthe out or pul genes derived from, respectively, Erwinia or Klebsiella.Moreover, the skilled artisan will appreciate that any secretoryprotein/s derived from a related host that is sufficiently homologous tothe out or pul gene/s described herein may also be employed (Pugsley etal., (1993) Microbiological Reviews 57:50-108; Lindeberg et al., (1996)Mol. Micro. 20:175-190; Lindeberg et al., (1992) J. of Bacteriology174:7385-7397; He et al., (1991) Proc. Natl. Acad. Sci. USA,88:1079-1083).

The term “derived from” is intended to include the isolation (in wholeor in part) of a polynucleotide segment from an indicated source or thepurification of a polypeptide from an indicated source. The term isintended to include, for example, direct cloning, PCR amplification, orartificial synthesis from, or based on, a sequence associated with theindicated polynucleotide source.

The term “ethanologenic” is intended to include the ability of amicroorganism to produce ethanol from a carbohydrate as a primaryfermentation product. The term includes but is not limited to naturallyoccurring ethanologenic organisms, organisms with naturally occurring orinduced mutations, and organisms that have been genetically modified.

The term “Gram-negative bacteria” is intended to include the artrecognized definition of this term. Typically, Gram-negative bacteriainclude, for example, the family Enterobacteriaceae which comprises,among others, the species Escherichia and Klebsiella.

The term “sufficiently homologous” is intended to include a first aminoacid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent amino acid residues or nucleotides,e.g., an amino acid residue which has a similar side chain, to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences share common structural domains and/or acommon functional activity. For example, amino acid or nucleotidesequences which share common structural domains have at least about 40%homology, preferably 50% homology, more preferably 60%, 70%, 80%, or 90%homology across the amino acid sequences of the domains and contain atleast one, preferably two, more preferably three, and even morepreferably four, five, or six structural domains, are defined herein assufficiently homologous. Furthermore, amino acid or nucleotide sequenceswhich share at least 40%, preferably 50%, more preferably 60%, 70%, 80%,or 90% homology and share a-common functional activity are definedherein as sufficiently homologous.

In one embodiment, two polynucleotide segments, e.g., promoters, are“sufficiently homologous” if they have substantially the same regulatoryeffect as a result of a substantial identity in nucleotide sequence.Typically, “sufficiently homologous” sequences are at least 50%, morepreferably at least 60%, 70%, 80%, or 90% identical, at least in regionsknown to be involved in the desired regulation. More preferably, no morethan five bases differ. Most preferably, no more than five consecutivebases differ.

To determine the percent identity of two polynucleotide segments, or twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1-989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

The polynucleotide and amino acid sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences, e.g., promoter sequences. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.(1990) J. Mol Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to polynucleotide molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to polypeptide molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http:H/www.ncbi.nlm.nih.gov.

II. Synergism Between Endoglucanases

The present invention is based, at least in part, on the discovery thatendoglucanases act synergistically in degrading complex sugars. Thisinvention is based, also in part, on the functional integration andexpression of two endoglucanases (e.g., EGY and EGZ) by an ethanologenichost cell (e.g., K. oxytoca P2) to effect synergistic degradation ofoligosaccharides (e.g., crystalline cellulose) and increase theproduction of ethanol by simultaneous saccharification and fermentation(SSF).

In one embodiment, an endoglucanase is derived from E. chrysanthemi andis the endoglucanase EGZ, which is encoded by the celZ gene (Boyer, etal. (1987) Eur. J. Biochem. 162:311-316). In another embodiment, anendoglucanase is derived from E. chrysanthemi and is the endoglucanaseEGY, which is encoded by the celY gene (Guiseppi et al., (1991) Gene106:109-114). E. chrysanthemi EGY and EGZ are endoglucanases that havehigh activities in the degradation of carboxymethyl cellulose (CMC) andbelong to, respectively, Type IV and Type II secretion groups (Hueck etal. (1998) Micro and Mol Biol Rev 62:379-433). EGY and EGZ differ insubstrate range and function synergistically during the hydrolysis ofCMC and amorphous cellulose, indicating a potential need for bothenzymes for optimal cellulase activity. Specifically, EGZ hydrolyzescellotriose, cellotetraose, cellopentaose, amorphous cellulose, and CMC.EGY hydrolyses polymeric substrates to products of approximately 10glucosyl residues.

In another embodiment, the endoglucanases (e.g., EGY and EGZ) arepurified separately and combined in a ratio sufficient for thesynergistic degradation of an oligosaccharide substrate to occur andthis may be determined using the assays disclosed herein. These assaysallow for a determination and optimization of a ratio between, e.g., twogiven glucanases, e.g., endoglucanases. Typically the ratios range fromabout 9 to 1 to about 19 to 1. In one embodiment, the ratio can be about9 to 1 or 19 to 1 for EGZ to EGY. In a preferred embodiment, optimumsynergy is observed with a high ratio of EGZ to EGY, similar to thatproduced by E. chrysanthemi and by SZ21, a K. oxytoca recombinant whichexpresses celY and celZ (See example 4).

In yet another embodiment, the endoglucanases can be combinedconcurrently with the oligosaccharide substrate. In yet anotherembodiment, the endoglucanases can be added to the oligosaccharidesubstrate sequentially. In a preferred embodiment, EGZ is sequentiallyadded to the substrate following the addition of EGY, after heatinactivation of EGY activity. Example 3 describes, in detail, thesynergistic effect of various ratios of endoglucanases (e.g., EGY andEGZ), the synergistic effect of sequential addition of endoglucanases(e.g., EGY and EGZ) (See Table 12), and the effect of various substrateconcentrations (See Table 11) and incubation times on the synergisticactivity of the endoglucanases.

In yet another embodiment, the synergistic degradation of theoligosaccharide is of a factor of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or 2.0 with a more preferable factor being about 1.8. Thesynergistic factor is calculated as the observed degradation divided bythe sum of predicted contributions from EGY alone and EGZ alone (Riedel,et al. (1997) FEMS Microbiol. Lett. 147:239-243). In one embodiment, theendoglucasases are EGY and EGZ. The predicted contribution of EGY aloneis about 10% of the total activity, and the predicted contributions ofEGZ alone are about 90% of the total activity.

In another aspect of the invention, at least one of the endoglucanasesis derived from a cell extract. The cell may be recombinantly engineeredto produce at least one endoglucanase. In a preferred embodiment, thecell is recombinantly engineered to produce two endoglucanases (e.g.,EGY and EGZ). For example, the cell can be a bacterial cell. Therecombinant cell comprises at least one heterologous polynucleotidesegment, or, preferably, two or more heterolgous polynucleotidessegments, encoding a polypeptide/s under the transcriptional control ofone or more heterologous surrogate promoter/s. The heterologouspolynucleotide and surrogate promoter may be plasmid based or integratedinto the genome of the organism (as described in the examples). In apreferred embodiment, the host cell is used as a source of a desiredpolypeptide for use in the bioconversion of a complex sugar to ethanol,or a step thereof. In another preferred embodiment, the heterologouspolynucleotide segment encodes one or more endoglucanases (e.g., EGY orEGZ) which are expressed at higher levels than are naturally occurringin the host. In one embodiment, the endoglucanases are purifiedseparately from different recombinant cells and subsequently combined tosynergistically degrade a substrate (e.g., an oligosaccharide). Inanother embodiment, one recombinant host cell can produce two or moreendoglucanases concurrently and can act synergistically to degrade asubstrate.

In another aspect of the invention, the recombinant bacterial host cellis an ethanologenic bacterium such as, for example, K. oxytoca P2, anethanologic derivative of M5A1 (Wood, et al. (1992) Appl. Environ.Microbiol. 58:2103-2110). In one embodiment, the recombinantethanologenic bacterium contains at least one heterologouspolynucleotide segment (e.g., celY or celZ derived from Erwinia)encoding at least one endoglucanase (e.g., EGY or EGZ). In a preferredembodiment, the recombinant ethanologenic bacteria contains more thanone heterologous polynucleotide segments which encode endoglucanases.For example, as described in detail in Example 4, celY and celZ can befunctionally integrated, expressed, and secreted from the ethanologicstrain K. oxytoca P2 concurrently to produce ethanol from anoligosaccharide substrate (e.g., crystalline cellulose).

In another embodiment, the recombinant host is a Gram-negativebacterium. In yet another embodiment, the recombinant host is from thefamily Enterobacteriaceae. The ethanologenic hosts of U.S. Pat. No.5,821,093, hereby incorporated by reference, for example, are suitablehosts and include, in particular, E. coli strains KO4 (ATCC 55123), KO11(ATCC 55124), and KO12 (ATCC 55125), and Klebsiella oxytoca strain P2(ATCC 55307). Alternatively, a non-ethanologenic host of the presentinvention may be converted into an ethanologenic host (such as theabove-mentioned strains) by introducing, for example, ethanologenicgenes from an efficient ethanol producer like Zymomonas mobilis. Thistype of genetic engineering, using standard techniques, results in arecombinant host capable of efficiently fermenting sugar into ethanol.In addition, the LY01 ethanol tolerant strain (ATCC 11303) may beemployed as described in published PCT international application WO98/45425 and this published application is hereby incorporated byreference (see also, e.g., Yomano et al. (1998) J. of Ind. Micro. & Bio.20:132-138).

In another preferred embodiment, the invention makes use of anon-ethanologenic recombinant host, e.g., E. coli strain B, E. colistrain DH5α, or Klebsiella oxytoca strain M5A1. These strains may beused to express at least one desired polypeptide, e.g., anendoglucanase, using techniques described herein. In addition, theserecombinant hosts may be used in conjunction with another recombinanthost that expresses yet another desirable polypeptide, e.g., a differentendoglucanase. For example, a recombinant host producing EGZ can becombined with a recombinant host producing EGY to produce a synergisticeffect. In addition, the non-ethanologenic host cell/s may be used inconjunction with an ethanologenic host cell. For example, the use of anon-ethanologenic host/s for carrying out, e.g., the synergisticdepolymerization of a complex sugar may be followed by the use of anethanologenic host for fermenting the depolymerized sugar. Accordingly,it will be appreciated that these reactions may be carried out seriallyor contemporaneously using, e.g., homogeneous or mixed cultures ofnon-ethanologenic and ethanologenic recombinant hosts.

In a preferred embodiment, one or more genes for fermenting a sugarsubstrate into ethanol are provided on a plasmid or integrated into thehost chromosome. More preferably, genes for fermenting a sugar substrateinto ethanol, e.g., pyruvate decarboxylase (e.g., pdc) and/or alcoholdehydrogenase (e.g., adh) are introduced into the host of the inventionusing an artificial operon such as the PET operon as described in U.S.Pat. No. 5,821,093, hereby incorporated by reference. Indeed, it will beappreciated that the present invention, in combination with what isknown in the art, provides techniques and vectors for introducingmultiple genes into a suitable host (see, e.g., Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons (1992),Sambrook, J. et al., Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), and Bergey's Manual of DeterminativeBacteriology, Kreig et al., Williams and Wilkins (1984), herebyincorporated by reference).

Accordingly, using the methods of the invention, a single geneticconstruct can encode all of the necessary gene products (e.g., aglucanase, an endoglucanase, an exoglucanase, a secretory protein/s,pyruvate decarboxylase, alcohol dehydrogenase) for performingsimultaneous saccharification and fermentation (SSF). For example,Example 4 describes, in detail, the simultaneous saccharification andfermentation (SSF) of crystalline cellulose (Sigmacell 50) by bacterialcellulases EGY and EGZ produced by ethanologenic K. oxytoca, with addedcommercial cellulase (Spezyme®). The endoglucanases produced byethanologenic K. oxytoca and the commercial cellulase (Spezyme®)function synergistically to increase ethanol production (7% to 22%) fromcrystalline cellulose (Sigmacell 50). The beneficial effect isattributed almost exclusively to EGY, despite the fact that EGYactivities were low in comparison to EGZ. Activity of the ethanologenicK. oxytoca strain SZ22, which expresses EGY, was nearly equivalent tothe activity of the ethanologenic K. oxytoca strain SZ21, whichexpresses both EGY and EGZ activities. K. oxytoca strain SZ6, whichexpresses only EGZ showed little benefit from the production of over20,000 U of endoglucanase activity per liter.

In one embodiment, the composition of two endoglucanases which actsynergistically to degrade an oligosaccharide also includes at least oneadditional enzymatic activity. This additional activity may be aglucanase activity selected from the group consisting of endoglucanase,exoglucanase, cellobiohydrolase, β-glucosidase, endo-1,4-β-xylanase,α-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase,acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase,β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase,pectate lyase, or a combination thereof. In another embodiment, thisadditional enzymatic activity may be derived from a fungus, for exampleT. longibranchiatum. Fungi such as T. longibranchiatum produce multipleendoglucanase activities, which are presumed to function together withexoglucanases during the hydrolysis of crystalline cellulose (Nidetzky,et al. (1995) Synergistic interaction of cellulases from Trichodermareesei during cellulose degradation p. 90-112. In J. N. Saddler and M.E. Himmel (ed.). Enzymatic degradation of insoluble carbohydrates ACSsymposium series 618, American Chemical Society, Washington, D.C.;Tomme, et al. (1995) Adv. Microbiol. Physiol. 37:1-81; Woodward, J.(1991) Bioresource Technol. 36:67-75).

In contrast to EGZ, EGY does not hydrolyze soluble cellobiosides butpreferentially acts on longer chain substrates, producing ends, whichcan function as new sites for exoglucanase activity. In the absence offungal cellulase additions, EGY and EGZ function synergistically todegrade amorphous cellulose. In nature, lignocellulosic substrates aredepolymerized by mixtures of extracellular enzymes produced by consortiaof fungi and bacteria. Thus, a mixture of E. chrysanthemi enzymes andenzymes from the fungus T. reesei can improve the digestion oflignocellulosic substrates during bioconversion to ethanol.

It will also be appreciated that a recombinant host may be furthermanipulated, using methods known in the art, to have mutations in anyendogenous gene/s (e.g., recombinase genes) that would interfere withthe stability, expression, function, and secretion of the introducedgenes. Further, it will also be appreciated that the invention isintended to encompass any regulatory elements, gene/s, or gene products,i.e., polypeptides, that are sufficiently homologous to the onesdescribed herein.

For effective degradation of oligosaccharides, the glucanase (e.g., EGYor EGZ) is preferably secreted into the extracellular millieu.Accordingly, in another embodiment of the invention, the host cell hasbeen engineered to express a secretory protein/s to facilitate theexport of the desired polypeptide from the cell. In one embodiment, thesecretory protein or proteins are derived from a Gram-negative bacterialcell, e.g., a cell from the family Enterobacteriaceae. In anotherembodiment, the secretory protein/s are from Erwinia and are encoded bythe out genes. In another embodiment, the secretory proteins are the pulgenes derived from Klebsiella. The introduction of one or more of thesesecretory proteins is especially desirable if the host cell is anenteric bacterium, e.g., a Gram-negative bacterium having a cell wall.Representative Gram-negative host cells of the invention are from thefamily Enterobacteriaceae and include, e.g., Escherichia and Klebsiella.In one embodiment, the introduction of one or more secretory proteinsinto the host results in an increase in the secretion of the selectedprotein, e.g., a glucanase, as compared to naturally-occurring levels ofsecretion. Preferably, the increase in secretion is at least about 10%and more preferably, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, or more, as compared to naturally-occurring levels ofsecretion. In a preferred embodiment, the addition of secretion genesallows for the glucanase polypeptide to be produced at higher levels. Ina preferred embodiment, the addition of secretion genes allows for theglucanase polypeptide to be produced with higher enzymatic activity. Ina most preferred embodiment, the glucanase is produced at higher levelsand with higher enzymatic activity. Preferably, an increase in glucanaseactivity of at least about 10%, more preferably about 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% is observed. Most preferably, anincrease in glucanase activity of several fold is obtained, e.g., about200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, as compared tocells without secretion genes (e.g., cells that either lack or do notexpress secretion genes at a sufficient level). The techniques andmethods for introducing such genes and measuring increased output of adesired polypeptide such as, e.g., a glucanase, are described in furtherdetail in the examples. Other equivalent methods are known to thoseskilled in the art.

EGY and EGZ are secreted by different mechanisms. As described inExample 4, approximately 70% of the EGZ produced was secreted as anextracellular product when the E. chrysanthemi out genes were added on aplasmid (pCPP2006) consistent with a Type II secretion system (Hueck, C.J. (1998) Microbiol. Mol. Biol. Rev. 62:379-433). Half of the EGYactivity was secreted in the presence or absence of the out genesconsistent with a Type IV secretion system (Hueck, C. J. supra).

Methods for screening strains having the introduced genes (e.g.,endoglucanase encoding genes such as celY and celZ and alcoholdehydrogenase genes) are routine and may be facilitated by visualscreens that can identify cells expressing either the alcoholdehydrogenase (ADH) or glucanase (e.g., EGZ or EGY) gene product. TheADH gene product produces acetaldehyde that reacts with theleucosulfonic acid derivative of p-roseaniline to produce an intenselyred product. Thus, ADH-positive clones can be easily screened andidentified as bleeding red colonies. Methods for screening for apolysaccharase activity (e.g., an endoglucanase , such as EGZ or EGY),also results in a clear visual phenotype as described below and in theexamples.

Methods for screening for synergism between two endoglucanases aredescribed in detail in the examples. EGY and EGZ can be purifiedseparately from recombinant host cells transformed with a plasmidcontaining the celY gene or the celZ gene. The cell-free culture brothcan be used to determine extracellular endoglucanase activity. Brothcontaining cells disrupted by ultrasound can be used to determine totalactivity. Hydrolysis of cellooligosaccharides can be analyzed by thinlayer chromatography. Additionally, endoglucanase activity using CMC asa substrate can be determined in vitro by analyzing samples for reducingsugars. Reducing sugars can be measured using 3,5-dinitrosalicyclic acidreagent with glucose as a standard. Synergy is calculated as theobserved activity divided by the sum of predicted contributions from EGYalone (10%) plus EGZ alone (90%).

Alternatively, recombinant host cells can be transformed with a plasmidcontaining both endoglucanases to produce both endoglucanasesconcurrently. Synergy can be determined as described above. Simultaneoussaccharification and fermentation (SSF) can be carried out to observeethanol production from a substrate (as described in Example 4). In SSFexperiments with added commercial cellulase, two of the K. oxytocarecombinants containing at least one E. chrysanthemi endoglucanaseproduced more ethanol than the parent K. oxytoca strain which lacked theE. chrysanthemi endoglucanases. Both of these strains also producedethanol levels equivalent to the best yeast SSF experiments (Cho, et al.(1999) J. Microbiol. Biotechnol. 9:340-345) using approximatelyone-third of the amount of added commercial cellulase (500 FPU/gcellulose versus 18 FPU/g cellulose for recombinant yeast).

Recombinant bacteria expressing, for example, the PET operon typicallygrow to higher cell densities in liquid culture than the unmodifiedparent organisms due to the production of neutral rather than acidicfermentation products (Ingram et al., (1988) Appl. Environ. Microbiol.54:397-404). On plates, ethanologenic clones are readily apparent aslarge, raised colonies which appear much like yeast. These traits havebeen very useful during the construction of new strains and can providea preliminary indication of the utility of new constructs. Rapidevaluations of ethanol producing potential can also be made by testingthe speed of red spot development on aldehyde indicator plates (Conwayet al, (1987) J. Bacteriol. 169:2591-2597). Typically, strains whichprove to be efficient in sugar conversion to ethanol can be recognizedby the production of red spots on aldehyde indicator plates withinminutes of transfer.

In a most preferred embodiment of the invention, a single host cell isethanologenic, that is, has all the necessary genes, either naturallyoccurring or artificially introduced or enhanced (e.g., using asurrogate promoter and/or genes from a different species or strain),such that the host cell has the ability to produce and secrete twoglucanses, preferably, an endoglucanase, more preferably at least twoendoglucanases sufficient to, degrade a complex sugar, and ferment thedegraded sugar into ethanol. Accordingly, such a host is suitable forsimultaneous saccharification and fermentation.

Moreover, the present invention takes into account that the native E.coli fermentation pathways produce a mixture of acidic and neutralproducts (in order of abundance): lactic acid, hydrogen+carbon dioxide(from formate), acetic acid, ethanol, and succinate. However, the Z.mobilis PDC (pyruvate decarboxylase) has a lower Km for pyruvate thanany of the competing E. coli enzymes. By expressing high activities ofPDC, carbon flow is effectively redirected from lactic acid andacetyl-CoA into acetylaldehyde and ethanol. Small amounts of succinatecan be eliminated by deleting the fumarate reductase gene (frd) (Ingramet al., (1991) U.S. Pat. No., 5,000,000; Ohta et al., (1991) Appl.Environ. Microbiol. 57:893-900). Additional mutations (e.g., in the pflor ldh genes) may be made to completely eliminate other competingpathways (Ingram et al., (1991) U.S. Pat. No., 5,000,000). Additionalmutations to remove enzymes (e.g., recombinases, such as recA) that maycompromise the stability of the introduced genes (either plasmid-basedor integrated into the genome) may also be introduced, selected for, orchosen from a particular background.

In addition, it should be readily apparent to one skilled in the artthat the ability conferred by the present invention, to transform genescoding for a protein or an entire metabolic pathway into a singlemanipulable construct, is extremely useful. Envisioned in this regard,for example, is the application of the present invention to a variety ofsituations where genes from different genetic loci are placed on achromosome. This may be a multi-cistronic cassette under the control ofa single promoter or separate promoters may be used.

Exemplary E. coli strains that are ethanologenic and suitable forfurther improvement according to the methods of the invention include,for example, KO4, KO11, and KO12 strains, as well as the LY01 strain, anethanol-tolerant mutant of the E. coli strain KO11. Ideally, thesestrains may be derived from the E. coli strain ATCC 11303, which ishardy to environmental stresses and can be engineered to beethanologenic and secrete a polysaccharase/s. In addition, recent PCRinvestigations have confirmed that the ATCC 11303 strain lacks all genesknown to be associated with the pathogenicity of E. coli (Kuhnert etal., (1997) Appl. Environ. Microbiol. 63:703-709).

Another preferred ethanologenic host for improvement according to themethods of the invention is the E. coli KO11 strain which is capable offermenting hemicellulose hydrolysates from many differentlignocellulosic materials and other substrates (Asghari et al., (1996)J. Ind. Microbiol. 16:42-47; Barbosa et al., (1992) Current Microbiol.28:279-282; Beall et al., (1991) Biotechnol. Bioeng. 38:296-303; Beallet al., (1 992) Biotechnol. Lett. 14:857-862; Hahn-Hagerdal et al.,(1994) Appl. Microbiol. Biotechnol. 41:62-72; Moniruzzaman et al.,(1996) Biotechnol. Lett. 18:955-990; Moniruzzaman et al., (1998)Biotechnol. Lett. 20:943-947; Grohmann et al., (1994) Biotechnol. Lett.16:281-286; Guimaraes et al., (1992) Biotechnol. Bioeng. 40:41-45;Guimaraes et al., (1992) Biotechnol. Lett. 14:415-420; Moniruzzaman etal., (1997) J. Bacteriol. 179:1880-1886). In FIG. 1, the kinetics ofbioconversion for this strain are shown. In particular, this strain isable to rapidly ferment a hemicellulose hydrolysate from rice hulls(which contained 58.5 g/L of pentose sugars and 37 g/L of hexose sugars)into ethanol (Moniruzzaman et al., (1998) Biotechnol. Lett. 20:943-947).It was noted that this strain was capable of fermenting a hemicellulosehydrolysate to completion within 48 to 72 hours, and under idealconditions, within 24 hours.

Another preferred host cell of the invention is the bacteriumKlebsiella. In particular, Klebsiella oxytoca is preferred because, likeE. coli, this enteric bacterium has the native ability to metabolizemonomeric sugars, which are the constituents of more complex sugars.Moreover, K. oxytoca has the added advantage of being able to transportand metabolize cellobiose and cellotriose, the soluble intermediatesfrom the enzymatic hydrolysis of cellulose (Lai et al., (1996) Appl.Environ. Microbiol. 63:355-363; Moniruzzaman et al., (1997) Appl.Environ. Microbiol. 63:4633-4637; Wood et al., (1992) Appl. Environ.Microbiol. 58:2103-2110). The invention provides genetically engineeredethanologenic derivatives of K. oxytoca, e.g., strain M5A1 having the Z.mobilis pdc and adhB genes encoded within the PET operon (as describedherein and in U.S. Pat. No. 5,821,093; Wood et al, (1992) Appl. Environ.Microbiol. 58:2103-2110).

Accordingly, the resulting organism, strain P2, produces ethanolefficiently from monomer sugars and from a variety of saccharidesincluding raffinose, stachyose, sucrose, cellobiose, cellotriose,xylobiose, xylotriose, maltose, etc. (Burchhardt et al., (1992) Appl.Environ. Microbiol. 58:1128-1133; Moniruzzaman et al., (1997) Appl.Environ. Microbiol. 63:4633-4637; Moniruzzaman et al., (1997) J.Bacteriol. 179:1880-1886; Wood et al., (1992) Appl. Environ. Microbiol.58:2103-2110). These strains may be further modified according to themethods of the invention to express and secrete one or morepolysaccharases (e.g., endoglucanases). Accordingly, this strain issuitable for use in the bioconversion of a complex saccharide in an SSFprocess (Doran et al., (1993) Biotechnol. Progress. 9:533-538; Doran etal., (1994) Biotechnol. Bioeng. 44:240-247; Wood et al., (1992) Appl.Environ. Microbiol. 58:2103-2110). In particular, the use of thisethanologenic P2 strain eliminates the need to add supplementalcellobiase, and this is one of the least stable components of commercialfungal cellulases (Grohmann, (1994) Biotechnol. Lett. 16:281-286).

Screen for Promoters Suitable for Use in Heterologous Gene Expression

While in one embodiment, the surrogate promoter of the invention is usedto improve the expression of a heterologous gene, e.g., a polysaccharase(an endoglucanase for example), it will be appreciated that theinvention also allows for the screening of surrogate promoters suitablefor enhancing the expression of any desirable gene product. In general,the screening method makes use of the cloning vector described inExample 1 and depicted in FIG. 3 that allows for candidate promoterfragments to be conveniently ligated and operably-linked to a reportergene. In one embodiment, the celZ gene encoding glucanase serves as aconvenient reporter gene because a strong colorimetric change resultsfrom the expression of this enzyme (glucanase) when cells bearing theplasmid are grown on a particular media (CMC plates). Accordingly,candidate promoters, e.g., a particular promoter sequence or,alternatively, random sequences that can be “shotgun” cloned andoperably linked to the vector, can be introduced into a host cell andresultant colonies are scanned, visually, for having increased geneexpression as evidenced by a phenotypic glucanase-mediated colorimetricchange on a CMC plate. Colonies having the desired phenotype are thenprocessed to yield the transforming DNA and the promoter is sequencedusing appropriate primers (see Example 1 for more details).

The high correspondence between the glucanase-mediated calorimetricchange on a CMC plate and expression levels of the enzyme is anexcellent indication of the strength of a candidate promoter (FIG. 4).Hence, the methods of the invention provide a rapid visual test forrating the strength of candidate surrogate promoters. Accordingly,depending on the desired expression level needed for a specific geneproduct, a particular identified surrogate promoter can be selectedusing this assay. For example, if simply the highest expression level isdesired, then the candidate promoter that produces the largestcalorimetric change may be selected. If a lower level of expression isdesired, for example, because the intended product to be expressed istoxic at high levels or must be expressed at equivalent levels withanother product, a weaker surrogate promoter can be identified,selected, and used as described.

The plasmid pLOI2311 contains the celY coding region under the controlof the lac promoter and pLOI2316 was identified as a clone oriented toexpress celY from the lac promoter as determined by endoglucanaseindicator plates. Replacement of the native promoter with the lacpromoter increased celY expression by recombinant E. coli harboring thisplasmid. In addition, to minimize problems associated with theexpression of heterologous genes in industrial strains such as theethanologenic K. oxytoca P2 strain, unregulated promoters were isolatedfrom random fragments of Z. mobilis DNA using functional assays. Usingthis method, a plasmid containing a Z. mobilis Sau3A1 fragment as aheterolgous was constructed (pLOI2323) (see example 4 and FIGS. 13 and14). In addition, high levels of EGZ were produced by E. coli harboringthe plasmid pLOI1620. See examples 3 and 4 for more details concerningthe construction of the plasmids and selection of heterologouspromoters.

Example 4 describes the construction of the celY, celZ integrationvector with surrogate promoters (pLOI2352) (See FIG. 15). To ensure thestability of this plasmid, hybrid genes were integrated into thechromosome, and the antibiotic resistance markers used in constructionwere deleted using the FLP recombinase system (Martinez-Morales, et al.(1999) J. Bacteriol. 181:7143-7148) to facilitate further geneticmodifications.

III. Methods of Use

Degrading or Depolymerizing a Complex Saccharide

In one embodiment, the host cell of the invention is used to degrade ordepolymerize a complex sugar, e.g., lignocellulose or an oligosaccharideinto a smaller sugar moiety. To accomplish this, the host cell of theinvention preferably expresses one or more polysaccharases, e.g.,endoglucanases, such as EGY and EGZ and these polysaccharases may beliberated naturally from the producer organism. Alternatively, thepolysaccharase is liberated from the producer cell by physicallydisrupting the cell. Various methods for mechanically (e.g., shearing,sonication), enzymatically (e.g., lysozyme), or chemically disruptingcells, are known in the art, and any of these methods may be employed.Once the desired polypeptide is liberated from the inner cell space itmay be used to degrade a complex saccharide substrate into smaller sugarmoieties for subsequent bioconversion into ethanol. The liberatedpolysaccharase may be purified using standard biochemical techniquesknown in the art. Alternatively, the liberated polysaccharase need notbe purified or isolated from the other cellular components and can beapplied directly to the sugar substrate.

Accordingly, it will be appreciated by the skilled artisan that one ormore polysaccharases can be selected for their activity and acomposition may be formulated having an optimized activity, preferablysynergistic activity, for degrading a complex sugar. The composition maytake e.g., the form of an unpurified, semi-purified, or purifiedendoglucanase activity which is mixed with one or more endoglucanaseactivities in a ratio that provides optimal degrading of a complexsugar. Alternatively, each enzyme activity may be separately formulatedwith instructions for use, i.e., mixing or applying in a preferred orderand/or ratio, in order to achieve optimal degrading of a complex sugar.

In another embodiment, a host cell is employed that coexpresses one ormore polysaccharases and a secretory protein/s such that thepolysaccharases are secreted into the growth medium. This eliminates theabove-mentioned step of having to liberate the polysaccharases from thehost cell. When employing this type of host, the host may be useddirectly in an aqueous solution containing an oligosaccharide.

In another embodiment, a host cell of the invention is designed toexpress more than one polysaccharase or is mixed with another hostexpressing a different polysaccharase in a ratio sufficient for thesynergistic degradation of an oligosaccharide to occur. For example, onehost cell could express a heterologous endoglucanase (e.g., EGY) whileanother host cell could express another endoglucanase (e.g., EGZ), andthese cells could be combined to form a heterogeneous culture havingsynergistic activity in the degradation of oligosaccharides.Alternatively, in a preferred embodiment, a single host strain isengineered to produce all of the above polysaccharases. In either case,a culture of recombinant host/s is produced having high expression ofthe desired polysaccharases for application to an oligosaccharide. Ifdesired, this mixture can be combined with an additional cellulase,e.g., an exogenous cellulase, such as a fungal cellulase. This mixtureis then used to degrade a complex substrate. Alternatively, prior to theaddition of the complex sugar substrate, the polysaccharase/s arepurified from the cells and/or media using standard biochemicaltechniques and used as a pure enzyme source for depolymerizing a sugarsubstrate.

It will be appreciated by the skilled artisan, that theethanol-producing bacterial strains of the invention are superior hostsfor the production of recombinant proteins because, under anaerobicconditions (e.g., in the absence of oxygen), there is less opportunityfor improper folding of the protein (e.g., due to inappropriatedisulfide bond formation). Thus, the hosts and culture conditions of theinvention potentially result in the greater recovery of a biologicallyactive product.

Fermenting a Complex Saccharide

In a preferred embodiment of the present invention, the host cell havingthe above mentioned attributes is also ethanologenic. Accordingly, sucha host cell can be applied in synergistically degrading ordepolymerizing a complex saccharide into a monosaccharide. Subsequently,the cell can catabolize the simpler sugar into ethanol by fermentation.This process of concurrent complex saccharide depolymerization intosmaller sugar residues followed by fermentation is referred to assimultaneous saccharification and fermentation (SSF).

Typically, fermentation conditions are selected that provide an optimalpH and temperature for promoting the best growth kinetics of theproducer host cell strain and catalytic conditions for the enzymesproduced by the culture (Doran et al., (1993) Biotechnol. Progress.9:533-538). For example, for Klebsiella, e.g., the P2 strain, optimalconditions were determined to be between 35-37° C. and pH 5.0-pH 5.4.Under these conditions, even exogenously added fungal endoglucanases andexoglucanases are quite stable and continue to function for long periodsof time. Other conditions are discussed in the Examples. Moreover, itwill be appreciated by the skilled artisan, that only routineexperimentation is needed, using techniques known in the art, foroptimizing a given fermentation reaction of the invention.

Currently, the conversion of a complex saccharide such aslignocellulose, is a very involved, multi-step process. For example, thelignocellulose must first be degraded or depolymerized using acidhydrolysis. This is then followed by steps that separate liquids fromsolids and these products are subsequently washed and detoxified toresult in cellulose that can be further depolymerized (using addedcellulases) and finally, fermented by a suitable ethanologenic hostcell. In contrast, the fermenting of corn is much simpler in thatamylases can be used to break down the corn starch for immediatebioconversion by an ethanologenic host in essentially a one-stepprocess.

Accordingly, it will be appreciated by the skilled artisan that therecombinant hosts and methods of the invention afford the use of asimilarly simpler and more efficient process for fermentinglignocellulose. For example, the method of the invention is intended toencompass a method that avoids acid hydrolysis altogether. Moreover, thehosts of the invention have the following advantages, 1) efficiency ofpentose and hexose co-fermentation; 2) resistance to toxins; 3)production of enzymes for complex saccharide depolymerization; and 4)environmental hardiness. Therefore, the complexity of depolymerizinglignocellulose can be simplified using an improved biocatalyst of theinvention. Indeed, in one preferred embodiment of the invention, thereaction can be conducted in a single reaction vessel and in the absenceof acid hydrolysis, e.g., as an SSF process.

Potential Substrates for Bioconversion into Ethanol

One advantage of the invention is the ability to use a saccharide sourcethat has been, heretofore, underutilized. Consequently, a number ofcomplex saccharide substrates may be used as a starting source fordepolymerization and subsequent fermentation using the host cells andmethods of the invention. Ideally, a recyclable resource may be used inthe SSF process. Mixed waste office paper is a preferred substrate(Brooks et al., (1995) Biotechnol. Progress. 11:619-625; Ingram et al.,(1995) U.S. Pat. No. 5,424,202), and is much more readily digested thanacid pretreated bagasse (Doran et al., (1994) Biotech. Bioeng.44:240-247) or highly purified crystalline cellulose (Doran et al.(1993) Biotechnol. Progress. 9:533-538). Glucanases, both endoglucanasesand exoglucanases, contain a cellulose binding domain, and these enzymescan be readily recycled for subsequent fermentations by harvesting theundigested cellulose residue using centrifugation (Brooks et al., (1995)Biotechnol. Progress. 11:619-625). By adding this residue with boundenzyme as a starter, ethanol yields (per unit substrate) were increasedto over 80% of the theoretical yield with a concurrent 60% reduction infungal enzyme usage (FIG. 2). Such approaches work well with purifiedcellulose, although the number of recycling steps may be limited withsubstrates with a higher lignin content. Other substrate sources thatare within the scope of the invention include any type of processed orunprocessed plant material, e.g., lawn clippings, husks, cobs, stems,leaves, fibers, pulp, hemp, sawdust, newspapers, etc.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXAMPLE 1 Methods for Making Recombinant Escherichia Hosts Suitable forFermenting Oligosaccharides into Ethanol

In this example, methods for developing and using Escherichia hostssuitable for fermenting oligosaccharides into ethanol are described. Inparticular, a strong promoter is identified which can be used toincrease the expression of a polysaccharase (e.g., glucanase). Inaddition, genes from Erwinia chrysanthemi are employed to facilitatepolysaccharase secretion thereby eliminating the need for celldisruption in order to release the desired polysaccharase activity.

Throughout this example, the following materials and methods are usedunless otherwise stated.

Materials and Methods

Organisms and Culture Conditions

The bacterial strains and plasmids used in this example are listed inTable 1, below.

For plasmid constructions, the host cell E. coli DH5α was used. Theparticular gene employed encoding a polysaccharase (e.g., glucanase) wasthe celZ gene derived from Erwinia chrysanthemi P86021 (Beall, (1995)Ph.D. Dissertation, University of Florida; Wood et al., (1997) Biotech.Bioeng. 55:547-555). The particular genes used for improving secretionwere the out genes derived from E. chrysanthemi EC 16 (He et al., (1991)Proc. Natl. Acad. Sci. USA. 88:1079-1083).

Typically, host cell cultures were grown in Luria-Bertani broth (LB) (10g L⁻¹ Difco® tryptone, 5 g L⁻¹ Difco® yeast extract, 5 g L⁻¹ sodiumchloride) or on Luria agar (LB supplemented with 15 g L⁻¹ of agar). Forscreening host cells having glucanase celZ activity (EGZ), CMC-plates(Luria agar plates containing carboxymethyl cellulose (3 g L⁻¹)) wereused (Wood et al., (1988) Methods in Enzymology 160:87-112). Whenappropriate, the antibiotics ampicillin (50 mg L⁻¹), spectinomycin (100g L⁻¹), kanamycin (50 g L⁻¹) were added to the media for selection ofrecombinant or integrant host cells containing resistance markers.Constructs containing plasmids with a temperature conditional pSC101replicon (Posfai et al., (1997) J. Bacteriol. 179:4426-4428) were grownat 30° C. and, unless stated otherwise, constructs with pUC-basedplasmids were grown at 37° C. TABLE 1 Strains and Plasmids UsedStrains/Plasmids Description Sources/References Strains Z. mobilis CP4Prototrophic Osman, et al., (1985) J. Bact. 164:173-180 E. coli strainDH5α lacZ M15 recA Bethesda Research Laboratory E. coli strain BPrototrophic ATCC 11303 E. coli strain HB 101 RecA lacY recA ATCC 37159Plasmids pUC19 bla cloning vector New England Biolabs pST76-K kan lowcopy number, temp. sensitive Posfai, et al., (1997) J. Bacteriol.179:4426-4428 pRK2013 kan mobilizing helper plasmid (mob+) ATCC pCPP2006Sp^(r), ca. 40 kbp plasmid carrying the complete out He, et al,. (1991)P.N.A.S. genes from E. chrysanthemi EC16 88:1079-1083 pLOI1620 bla celZBeall, et al, (1995) Ph.D. Dissertation, U. of Florida pLOI2164 pLOI1620with BamHI site removed (Klenow) See text pLOI2170 NdeI-HindIII fragment(promoterless celZ) from See text pLOI2164 cloned into pUC19 pLOI2171BamHI-SphI fragment (promoterless celZ) from See text pLOI2170 clonedinto pST76-K pLOI2173 EcoRI-SphI fragment (celZ with native promoter)See text from pLOI2164 cloned into pST76-K pLOI2174 EcoRI-BamHI fragment(gap promoter) cloned into See text pLOI2171 pLOI2175 EcoRI-BamHIfragment (eno promoter) cloned into See text pLOI2171 pLOI2177 RandomSau3A1 Z. mobilis DNA fragment cloned See text into pLOI2171 pLOI2178Random Sau3A1 Z. mobilis DNA fragment cloned See text into pLOI2171pLOI2179 Random Sau3A1 Z. mobilis DNA fragment cloned See text intopLOI2171 pLOI2180 Random Sau3A1 Z. mobilis DNA fragment cloned See textinto pLOI2171 pLOI2181 Random Sau3A1 Z. mobilis DNA fragment cloned Seetext into pLOI2171 pLOI2182 Random Sau3A1 Z. mobilis DNA fragment clonedSee text into pLOI2171 pLOI2183 Random Sau3A1 Z. mobilis DNA fragmentcloned See text into pLOI2171 pLOI2184 Random Sau3A1 Z. mobilis DNAfragment cloned See text into pLOI2171 pLOI2196 pLOI2177 fused intopUC19 at the PstI site See text pLOI2197 pLOI2180 fused into pUC19 atthe PstI site See text pLOI2198 pLOI2182 fused into pUC19 at the PstIsite See text pLOI2199 pLOI2183 fused into pUC19 at the PstI site Seetext pLOI2307 EcoRI-SphI fragment from pLOI2183 cloned into See textpUC19Genetic Methods

Standard techniques were used for all plasmid constructions (Ausubel etal., (1987) Current Protocols in Molecular Biology, John Wiley & Sons,Inc.; Sambrook et al., (1989) Molecular cloning: a laboratory manual,2^(nd) ed. C. S. H. L., Cold Spring Harbor, N.Y). For conductingsmall-scale plasmid isolation, the TELT procedure was performed. Forlarge-scale plasmid isolation, the Promega® Wizard Kit was used. Forisolating DNA fragments from gels, the Qiaquick® Gel Extraction Kit fromQiagen® was employed. To isolate chromosomal DNA from E. coli and Z.mobilis the methods of Cutting and Yomano were used (Cutting et al.,(1990), Genetic analysis, pp. 61-74, In, Molecular biological methodsfor Bacillus, John Wiley & Sons, Inc.; Yomano et al., (1993) J.Bacteriol. 175:3926-3933).

To isolate the two glycolytic gene promoters (e.g., gap and eno)described herein, purified chromosomal DNA from E. coli DH5α was used asa template for the PCR (polymerase chain reaction) amplification ofthese nucleic acids using the following primer pairs: gap promoter,5′-CGAATTCCTGCCGAAGTTTATTAGCCA-3′ (SEQ ID NO: 3) and5′-AAGGATCCTTCCACCAGCTATTTGTTAGTGA-3′ (SEQ ID NO: 4); eno promoter,5′-AGAATTCTGCCAGTTGGTTGACGATAG-3′ (SEQ ID NO: 5) and5′-CAGGATCCCCTCAAGTCACTAGTTAAACTG-3′ (SEQ ID NO: 6). The out genesencoding secretory proteins derived from E. chrysanthemi (pCPP2006) wereconjugated into E. coli using pRK2013 for mobilization (Figurski et al.,(1979) Proc. Natl. Acad. Sci. USA. 7.6: 1648-1652; Murata et al., (1990)J. Bacteriol. 172:2970-2978).

To determine the sequence of various DNAs of interest, the dideoxysequencing method using fluorescent primers was performed on a LI-CORModel 4000-L DNA Sequencer. The pST76-K-based plasmids were sequenced inone direction using a T7 primer (5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO:7)). The pUC18- and pUCI9-based plasmids were sequenced in twodirections using either a forward primer (5′-CACGACGTTGTAAAACGAC-3′ (SEQID NO: 8)) or a reverse primer (5′-TAACAATTTCACACAGGA-3′ (SEQ ID NO:9)). The extension reactions of the sequencing method were performedusing a Perkin Elmer GeneAmp′ PCR 9600 and SequiTherm Long-ReadSequencing Kit-LC®. Resultant sequences were subsequently analyzed usingthe Wisconsin Genetic Computer Group (GCG) software package (Devereux etal., (1984) Nucleic Acids Rev. 12:387-395).

To determine the start of transcriptional initiation in theabove-mentioned promoters, primer extension analysis was performed usingstandard techniques. In particular, promoter regions were identified bymapping the transcriptional start sites using a primer findingcorrespondence within the celZ gene RNA that was isolated from cells inlate exponential phase using a Qiagen RNeasy® kit. Briefly, cells weretreated with lysozyme (400 μg/ml) in TE (Tris-HCl, EDTA) containing 0.2M sucrose and incubated at 25° C. for 5 min prior to lysis. LiberatedRNA was subjected to ethanol precipitation and subsequently dissolved in20 μl of Promega® AMV reverse transcriptase buffer (50 mM Tris-HCl, pH8.3, 50 mM KCl, 10 mM MgCl₂, 0.5 mM spermadine, 10 mM DTT). AnIRD41-labeled primer (5′-GACTGGATGGTTATCCGAATAAGAGAGAGG-3′ (SEQ ID NO:10)) from LI-Cor Inc. was then added and the sample was denatured at 80°C. for 5 min, annealed at 55° C. for 1 hr, and purified by alcoholprecipitation. Annealed samples were dissolved in 19 μl of AMV reversetranscriptase buffer containing 500 μM dNTPs and 10 units AMV reversetranscriptase, and incubated for extension (1 h at 42° C.). Productswere treated with 0.5 μg/ml DNase-free RNase A, precipitated, dissolvedin loading buffer, and compared to parallel dideoxy promoter sequencesobtained using the LI-COR Model 4000-L DNA sequencer.

Polysaccharase Activity

To determine the amount of polysaccharase activity (e.g., glucanaseactivity) resulting from expression of the celZ gene, a Congo Redprocedure was used (Wood et al., (1988) Methods in Enzymology160:87-112). In particular, selected clones were transferred to griddedCMC plates and incubated for 18 h at 30° C. and then stained andrecombinant host cells expressing glucanase formed yellow zones on a redbackground. Accordingly, the diameters of these colorimetric zones wererecorded as a relative measure of celZ expression.

Glucanase activity (EGZ) was also measured using carboxymethyl celluloseas a substrate. In this test, appropriate dilutions of cell-free culturebroth (extracellular activity) or broth containing cells treated withultrasound (total activity) were assayed at 35° C. in 50 mM citratebuffer (pH 5.2) containing carboxymethyl cellulose (20 g L⁻¹).Conditions for optimal enzyme release for 3-4 ml samples were determinedto be 4 pulses at full power for 1 second each using a cell disruptor(Model W-220F, Heat System-Ultrasonics Inc., Plainview, N.Y.). To stopthe enzyme reactions of the assay, samples were heated in a boilingwater bath for 10 min. To measure reducing sugars liberatedenzymatically by the glucanase, a dinitrosalicylic acid reagent wasemployed using glucose as a standard (Wood et al., (1988) Methods inEnzymology 160:87-112). The amount of enzyme activity (IU) was expressedas ,umols of reducing sugar released per min or as a percentage of totalactivity from an average of two or more determinations.

Ultrastructural Analysis

To determine the ultrastructure of various recombinant host cells, freshcolonies from Luria agar plates were prepared for analysis by fixing in2% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7) followed byincubation in 1% osmium tetroxide and followed by 1% uranyl acetate indistilled water. Samples were dehydrated in ethanol, embedded in Spurr'splastic, and ultrathin sections were prepared and examined using aZeiss® EM-IOCA electron microscope (Spur (1969) J. Ultrastruct. Res.26:31).

Construction of a Low Copy Promoter Probe Vector Using celZ as theReporter Gene

To facilitate the isolation of strong promoters, a low copy vector wasconstructed with a pSC101 replicon and a BamHI site immediatelypreceding a promoterless celZ gene (pL012171). Accordingly, thispromoterless plasmid was used as a negative control. The plasmidpLOI1620 was used as a source of celZ and is a pUC18 derivative withexpression from consecutive lac and celZ promoters. The BamHI site inthis plasmid was eliminated by digestion and Klenow treatment(pLO12164). The celZ gene was isolated as a promoterless NdeI fragmentafter Klenow treatment. The resulting blunt fragment was digested withHindIII to remove downstream DNA and ligated into pUC19 (HindIII toHincII) to produce pLOI2170. In this plasmid, celZ is oriented oppositeto the direction of lacZ transcription and was only weakly expressed.The BamHI (amino terminus)-SphI (carboxyl terminus) fragment frompLO12170 containing celZ was then cloned into the corresponding sites ofpST76-K, a low copy vector with a temperature sensitive replicon, toproduce pLOI2171 (FIG. 3). Expression of celZ in this vector wasextremely low facilitating its use as a probe for candidate strongpromoters.

Analysis of celZ Expression from Two E. coli Glycolytic Promoters (gapand eno)

Two exemplary promoters driving glycolytic genes (gap and eno) in E.coli were examined for their ability to drive the expression of theheterologous celZ gene encoding glucanase. Chromosomal DNA from the E.coli DH5α strain was used as a template to amplify the gap and enopromoter regions by the polymerase chain reaction. The resultingfragments of approximately 400 bp each were digested with EcoRI andBamHI and cloned into the corresponding sites in front of a promoterlesscelZ gene in pLO12171 to produce pLO12174 (gap promoter) and pLOI2175(eno promoter). As a control, the EcoRI-SphI fragment from pLOI2164containing the complete celZ gene and native E. chrysanthemi promoterwas cloned into the corresponding sites of pST76-K to produce pLOI2173.These three plasmids were transformed into E. coli strains B and DH5αand glucanase activity (EGZ) was compared. For both strains of E. coli,glucanase activities were lower on CMC plates with E. coli glycolyticpromoters than with pLO12173 containing the native E. chrysanthemipromoter (Table 2). Assuming activity is related to the square of theradius of each zone (Fick's Law of diffusion), EGZ production withglycolytic promoters (pLOI2174 and pLOI2175) was estimated to be 33% to65% lower than in the native promoter construct (pLO12373). Accordingly,other candidate promoters for driving high levels of celZ geneexpression were investigated.

Identifying and Cloning Random DNA Fragments Suitable for Use asPromoters for Heterologous Gene Expression

Random fragments derived from Z. mobilis can be an effective source ofsurrogate promoters for the high level expression of heterologous genesin E. coli. (Conway et al., (1987) J. Bacteriol. 169:2327-2335; Ingramet al., (1988) Appl. Environ. Micro. 54:397-404). Accordingly, toidentify surrogate promoters for Erwinia celZ expression, Z. mobilischromosomal DNA was extensively digested with Sau3AI and resultingfragments were ligated into pLOI2171 at the BamHI site and transformedinto E. coli DH5α to generate a library of potential candidatepromoters. To rapidly identify superior candidate promoters capable ofdriving celZ gene expression in E. coli, the following biological screenwas employed. Colonies transformed with celZ plasmids having differentrandom candidate promoters were transferred to gridded CMC plates andstained for glucanase activity after incubation (Table 2). Approximately20% of the 18,000 clones tested were CMC positive. The 75 clones whichproduced larger zones than the control, pLOI2173, were examined furtherusing another strain, E. coli B. TABLE 2 Evaluation of promoter strengthfor celZ expression in E. coil using CMC indicator plates. E. coli DH5αhost E. coli B host % of native Number % of native Number of CMC zonepromoter of CMC zone promoter Plasmids Plasmids^(a) diameter (mm)^(b)(100*R² _(x)/R² _(c))^(c) plasmids diameter (mm) (100*R² _(x)/R²c)pLOI217l 1 0 — — — — (promoterless) pLOI2173 1 5.0 100 1 4.5 100 (nativepromoter) pLOI2174 1 4.0 77 1 3.5 60 (gap promoter) pLOI2175 1 3.0 43 12.8 35 (eno promoter) Z. mobilis promoters Group I 5 13.0 676 410.8-11.3 570-625 Group II 14  9.0-11.0 324-484 17  9.0-10.5 445-545Group III 56 6.0-9.0 144-324 54 5.0-8.8 125-375^(a)The number of clones which the indicated range of activities.^(b)The average size of the diameters from three CMC digestion zones.^(c)R²x is the square of the radius of the clear zone with the testplasmid; R2c is the square of the radius of the clear zone for thecontrol (pLOI2173).

Thus, promoter strength for selected candidate promoters was confirmedin two different strains with, in general, recombinants of DH5αproducing larger zones (e.g., more glucanase) than recombinants ofstrain B. However, relative promoter strength in each host was similarfor most clones. Based on these analyses of glucanase production asmeasured by zone size using CMC plates, four clones appeared to expresscelZ at approximately 6-fold higher levels than the construct with theoriginal E. chrysanthemi celZ gene (pLO12173), and at 10-fold higherlevels than either of the E. coli glycolytic promoters. Accordingly,these and similarly strong candidate promoters were selected for furtherstudy.

Production and Secretion of Glucanase

Eight plasmid derivatives of pST76-K (pLOI2177 to pLOI2184) wereselected from the above-described screen (see Group I and Group II(Table 2)) and assayed for total glucanase activity in E. coli strain B(Table 3). The four plasmids giving rise to the largest zones on CMCplates were also confirmed to have the highest glucanase activities(pLOI2177, pLOI2180, pLOI2182, and pLOI2183). The activities wereapproximately 6-fold higher than that of the unmodified celZ (pLOI2173),in excellent agreement with our estimate using the square of the radiusof the cleared zone on CMC plates. FIG. 4 shows a comparison of activityestimates from CMC plates and in vitro enzyme assays for strain Bcontaining a variety of different promoters, with and without theaddition of out genes encoding secretory proteins. Although there issome scatter, a direct relationship is clearly evident which validatesthe plate method for estimating relative activity. The originalconstruct in pUC18, a high copy plasmid, was also included forcomparison (pLOI2164). This construct with consecutive lac and celZpromoters produced less EGZ activity than three of the low copy plasmidswith surrogate promoters (pLOI2177, pLOI2182, and pLOI2183). Thus, toincrease celZ expression of glucanase even more, the DNA fragmentcontaining celZ and the most effective surrogate promoter was isolatedfrom pLOI2183 (as a EcoRI-SphI fragment) and inserted into pUCl9 withtranscription oriented opposite to that of the lac promoter (pLOI2307).Accordingly, the above-identified strong surrogate promoter whenincorporated into a high copy plasmid, further increased glucanaseactivity by 2-fold.

Engineering Increased Secretion of Glucanase

To further improve on the above-described results for increasingexpression of celZ encoded glucanase, the above host cells wereengineered for increased secretion. Genes encoding secretory proteins(e.g., the out genes) derived from E. chrysanthemi EC16 were used forimproving the export of the glucanase using the plasmid as described inHe et al. that contains out genes (pCPP2006) (He et al., (1991) Proc.Natl. Acad. Sci. USA. 88:1079-1083). The increased secretion of EGZ inE. coli B was investigated and results are presented in Table 3. TABLE 3Comparison of promoters for EGZ production and secretion in E. coli BWith secretion genes (pCPP2006) Without secretion genesExtracellular^(c) Plasmids^(a) Total activity (IU/L)^(b)Extracellular^(c)(%) Total Activity (IU/L) (%) pLOI2173 620 17 1,100 43pLOI2177 3,700 10 5,500 44 pLO12178 2,200 9 3,500 49 pLOI2179 2,000 103,000 50 pLOI2180 2,900 8 6,300 39 pLOI2181 1,800 11 4,100 46 pLOI21823,500 7 6,600 38 pLOI2183 3,400 7 6,900 39 pLOI2184 2,100 12 2,400 39pLOI2164 3,200 20 6,900 74 pLOI2307 6,600 28 13,000 60^(a)Plasmids pLOI2173 and pLOI2164 contain the celZ native promoter;pLOI2307 contains the strong promoter from pLOI2183.Plasmids pLOI2164 and pLOI2307 are pUC-based plasmids (high copynumber). All other plasmids are derivatives of pST76-K (low copynumber).^(b)Glucanase activities were determined after 16 h of growth at 30° C.^(c)Extracellular activity (secreted or released).

Recombinant hosts with low copy plasmids produced only 7-17% of thetotal EGZ extracellularly (after 16 hours of growth) without theadditional heterologous secretory proteins (out proteins encoded byplasmid pCPP2006). A larger fraction of EGZ (20-28%) was found in theextracellular broth surrounding host cells with the high-copy pUC-basedplasmids than with the low copy pST76-based plasmids containing the samepromoters. However, in either case, the addition of out genes encodingsecretory proteins (e.g., pCPP2006) increased the total level ofexpression by up to 2-fold and increased the fraction of extracellularenzyme (38-74%) by approximately 4-fold. The highest activity, 13,000IU/L of total glucanase of which 7,800 IU/L was found in the cell-freesupernatant was produced by strain B having both pLOI2307 encoding celZdriven by a strong surrogate promoter and pCPP2006 encoding outsecretory proteins).

It has been reported that under certain conditions (pH 7, 370 C), thespecific activity for pure EGZ enzyme is 419 IU (Py et al., (1991)Protein Engineering 4:325-333) and it has been determined that EGZproduced under these conditions is 25% more active than under theabove-mentioned conditions (pH 5.2 citrate buffer, 35° C.). Accordingly,assuming a specific activity of 316 IU for pure enzyme at pH 5.2 (35°C.), the cultures of E. coli B (containing pLOI2307 and pCPP2006, e.g.,plasmids encoding glucanase and secretory proteins), producedapproximately 41 mg of active EGZ per liter or 4-6% of the total hostcell protein was active glucanase.

Sequence Analysis of the Strongest Promoter Derivedfrom Z. mobilis

The sequences of the four strongest surrogate promoters (pLOI2177,pLOI2180, pLOI2182, and pLOI2183) were determined. To facilitate thisprocess, each was fused with pUC19 at the PstI site. The resultingplasmids, pLOI2196, pLOI2197, pLOI2198, and pLO12199, were produced athigh copy numbers (ColEI replicon) and could be sequenced in bothdirections using M13 and T7 sequencing primers. All four plasmidscontained identical pieces of Z. mobilis DNA and were siblings. Each was1417 bp in length and contained 4 internal Sau3AI sites. DNA andtranslated protein sequences (six reading frames) of each piece werecompared to the current data base. Only one fragment (281 bp internalfragment) exhibited a strong match in a BLAST search (National Centerfor Biotechnology Information; http://www.ncbi.nlm.nih.gov/BLAST/) andthis fragment was 99% identical in DNA sequence to part of the Z.mobilis hpnB gene which is proposed to function in cell envelopebiosynthesis (Reipen et al., (1995) Microbiology 141:155-161). Primerextension analysis revealed a single major start site, 67 bp upstreamfrom the Sau3AI/BamHI junction site with celZ and a second minor startsite further upstream (FIG. 5). Sequences in the −10 and −35 regionswere compared to the conserved sequences for E. coli sigma factors (Wanget al., (1989) J. Bacteriol. 180:5626-5631; Wise et al., (1996) J.Bacteriol. 178:2785-2793). The dominant promoter region (approximately85% of total start site) appears similar to a sigma⁷⁰ promoter while thesecondary promoter site resembles a sigma³⁸ promoter.

Microscopic Analysis of Recombinant Host Cells Producing Glucanase

Little difference in cell morphology was observed between recombinantsand the parental organism by light microscopy. Under the electronmicroscope, however, small polar inclusion bodies were clearly evidentin the periplasm of strain B (pLOI2164) expressing high amounts ofglucanase and these inclusion bodies were presumed to contain EGZ (FIG.6). In the strain B (pLOI2307) that produced 2-fold higher glucanaseactivity the inclusion bodies were even larger and occupied up to 20% ofthe total cell volume. The large size of these polar bodies suggeststhat glucanase activity measurements may underestimate the total EGZproduction. Typically, polar inclusion bodies were smaller in host cellsalso having constructs encoding the out secretory proteins, which allowfor increased secretion of proteins from the periplasmic space. Asexpected, no periplasmic inclusion bodies were evident in the negativecontrol strain B (pUC19) which does not produce glucanase.

EXAMPLE 2 Recombinant Klebsiella Hosts Suitable for FermentingOligosaccharides into Ethanol

In this example, a recombinant Klebsiella host, suitable for use as abiocatalyst for depolymerizing and fermenting oligosaccharides intoethanol, is described.

Throughout this example, the following materials and methods are usedunless otherwise stated.

Materials and Methods

Bacteria, Plasmids, and Culture Conditions

The strains and plasmids that were used in this exemplification aresummarized in Table 4 below. TABLE 4 Strains and Plasmids UsedStrains/Plasmids Properties Sources/References Strains Zymomonas mobilisPrototrophic Ingram et al. (1988) Appl. CP4 Environ. Micro. 54:397-404Escherichia coli DH5α lacZ M15 recA Bethesda Research Laboratory HB101recA lacY recA ATCC 37159 Klebsiella oxytoca M5A1 Prototrophic Wood etal. (1992) Appl. Environ. Micro. 58:2103-2110 P2 Pfl::pdc adhB cat Woodet al. (1992) Appl. Environ. Micro. 58:2103-2110 SZ1 pfl::pdc adhB cat;integrated celZ; tet See text SZ2 pfl::pdc adhB cat; integrated celZ;tet See text SZ3 pfl::pdc adhB cat; integrated celZ; tet See text SZ4pfl::pdc adhB cat; integrated celZ; tet See text SZ5 pfl::pdc adhB cat;integrated celZ; tet See text SZ6 pfl::pdc adhB cat; integrated celZ;tet See text SZ7 pfl::pdc adhB cat; integrated celZ; tet See text SZ8pfl::pdc adhB cat; integrated celZ; tet See text SZ9 pfl::pdc adhB cat;integrated celZ; tet See text SZ10 pfl::pdc adhB cat; integrated celZ;tet See text Plasmids pUC19 bla cloning vector New England BiolabspBR322 bla tet cloning vector New England Biolabs pLOI1620 bla celZ Woodet al. (1997) Biotech. Bioeng. 55:547-555 pRK2013 kan mobilizing helperplasmid (mob⁺) ATCC pCPP2006 Sp^(r), 40 kbp fragment containing outgenes He et al. (1991) P.N.A.S. from E. chrysanthemi EC16 88:1079-1083pST76-K kan low copy vector containing temperature Posfai et al. (1997)J. Bact. sensitive pSC101 replicon 179:4426-4428 pLOI2164 bla celZ(BamHI eliminated from See text pLOI1620) pLOI2173 kan celZ (native celZpromoter) See text pLOI2177 kan celZ (surrogate promoter from Z. Seetext mobilis) pLOI2178 kan celZ (surrogate promoter from Z. See textmobilis) pLOI2179 kan celZ (surrogate promoter from Z. See text mobilis)pLOI2180 kan celZ (surrogate promoter from Z. See text mobilis) pLOI2181kan celZ (surrogate promoter from Z. See text mobilis) pLOI2182 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2183 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2184 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2185 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2186 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2187 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2188 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2189 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2190 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2191 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2192 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2193 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2194 kan celZ(surrogate promoter from Z. See text mobilis) pLOI2301 AscI linkerinserted into NdeI site of pUC19 See text pLOI2302 AscI linker insertedinto SapI site of See text pLOI2301 pLOI2303 AvaI-EcoRI fragment frompBR322 See text inserted into PstI site of pLOI2302 after Klenowtreatment pLO12305 EcoRI DNA fragment of K. oxytoca M5A1 See textgenomic DNA (Ca. 2.5 kb) cloned into the SmaI site of pLOI2303 pLO12306EcoRI-SphI fragment from pLOI2183 See text cloned into EcoRI site ofpLOI2305

The culture conditions used for cultivating E. coli and K. oxytoca M5A1typically employed Luria-Bertani broth (LB) containing per liter: 10 gDifco® tryptone, 5 g yeast extract, and 5 g sodium chloride, or,alternatively, Luria agar (LB supplemented with 15 g of agar) (Sambrooket al., (1989), Molecular Cloning: A Laboratory Manual, C. S. H. L.,Cold Spring Harbor, N.Y.).

For screening bacterial colonies under selective conditions, CMC-plates(Luria agar plates containing 3 g L⁻¹ carboxymethyl cellulose) were usedto determine levels of glucanase activity expressed by a given bacterialstrain (Wood et al. (1988) Enzymology, 160:87-112). For cultivatingethanologenic strains, glucose was added to solid media (20 g L⁻¹) andbroth (50 g L⁻¹). In determining glucanase activity, the glucose in thegrowth media was replaced with sorbitol (50 g L⁻¹), a non-reducingsugar. For cultivating various strains or cultures in preparation forintroducing nucleic acids by electroporation, a modified SOC medium wasused (e.g., 20 g L⁻¹ Difcoo tryptone, 5 g L⁻¹, Difcoo yeast extract, 10mM NaCl, 2.5 mM KCl, 10 mM MgSO₄, 10 mM MgCl₂, and 50 g L⁻¹ glucose).The antibiotics ampicillin (50 mg L⁻¹), spectinomycin (100 mg L⁻¹),kanamycin (50 mg L⁻¹), tetracycline (6 or 12 mg L⁻¹), andchloramphenicol (40, 200, or 600 mg L⁻¹) were added when appropriate forselection of recombinant hosts bearing antibiotic resistance markers.Unless stated otherwise, cultures were grown at 37° C. Ethanologenicstrains and strains containing plasmids with a temperature-sensitivepSC101 replicon were grown at 30° C.

Genetic Methods

For plasmid construction, cloning, and transformations, standard methodsand E. coli DH5α hosts were used (Ausubel et al. (1987) CurrentProtocols in Molecular Biology. John Wiley & Sons, Inc.; Sambrook etal., (1989) Molecular Cloning: A Laboratory Manual, C. S. H. L., ColdSpring Harbor, N.Y.). Construction of the celZ integration vector,pLOI2306, was performed as shown in FIG. 7. A circular DNA fragmentlacking a replicon from pLOI2306 (see FIG. 7) was electroporated intothe ethanologenic K. oxytoca P2 using a Bio-Rad Gene Pulser using thefollowing conditions: 2.5 kV and 25 μF with a measured time constant of3.8-4.0 msec (Comaduran et al. (1998) Biotechnol. Lett. 20:489-493). TheE. chrysanthemi EC 16 secretion system (pCPP2006) was conjugated into K.oxytoca using pRK2013 for mobilization (Murata et al. (1990) J.Bacteriol. 172:2970-2978). Small scale and large scale plasmidisolations were performed using the TELT procedure and a Promega WizardKit, respectively. DNA fragments were isolated from gels using aQiaquick® Gel Extraction Kit from Qiagen® (Qiagen Inc., Chatsworth,Calif.). Chromosomal DNA from K. oxytoca M5A1 and Z. mobilis CP4 wereisolated as described by Cutting and Yomano (see Example 1). The DNAs ofinterest were sequenced using a LI-COR Model 4000-L DNA sequencer (Woodet al. (1997) Biotech. Bioeng. 55:547-555).

Chromosomal Integration of celZ

Two approaches were employed for chromosomal integration of celZ, usingselection with a temperature-conditional plasmid (pLOI2183) using aprocedure previously described for E. coli (Hamilton et al., (1989) J.Bacteriol. 171:4617-4622) and direct integration of circular DNAfragments lacking a functional replicon. This same method was employedfor chromosomal integration of Z. mobilis genes encoding the ethanolpathway in E. coli B (Ohta K et al., (1991) Appl. Environ. Microbiol.57:893-900) and K. oxytoca M5A1 (Wood et al. (1992) Appl. Environ.Microbiol. 58:2103-2110). Typically, circular DNA was transformed intoP2 by electroporation using a Bio-Rad Gene Pulser. Next, transformantswere selected on solid medium containing tetracycline (6 mg L⁻¹) andgrown on CMC plates to determine levels of glucanase activity.

Glucanase Activity

Glucanase activity resulting from expression of celZ gene product (i.e.,glucanase) under the control of different test promoters was evaluatedby staining CMC plates as described in Example 1. This colorimetricassay results in yellow zones indicating glucanase activity and thediameter of the zone was used as a relative measure of celZ polypeptideexpression. Clones that exhibited the largest zones of yellow color werefurther evaluated for glucanase activity at 35° C. using carboxymethylcellulose as the substrate (20 g L⁻¹ dissolved in 50 mM citrate buffer,pH 5.2) (Wood et al. (1988) Methods in Enzymology 160: 87-112). In orderto measure the amount of intracellular glucanase, enzymatic activity wasreleased from cultures by treatment with ultra-sound for 4 seconds(Model W-290F cell disruptor, Heat System-Ultrasonics Inc., Plainview,N.Y.). The amount of glucanase activity expressed was measured and ispresented here as μmol of reducing sugar released per min (IU). Reducingsugar was measured as described by Wood (Wood et aL (1988) Methods inEnzymology 160: 87-112) using a glucose standard.

Substrate Depolymerization

To further determine the amount of glucanase activity produced byvarious host cells, different carbohydrate substrates (20 g L⁻¹suspended in 50 mM citrate buffer, pH 5.2) were incubated with variouscell extracts. In one example, test substrates comprising acid-swollenand ball-milled cellulose were prepared as described by Wood (Wood et aL(1988) Methods in Enzymology 160: 87-112). A typical polysaccharaseextract (i.e., EGZ (glucanase) from K. oxytoca SZ6 (pCPP2006)) wasprepared by cultivating the host cells at 30° C. for 16 h in LBsupplemented with sorbitol, a nonreducing sugar. Dilutions of cell-freebroth were added to substrates and incubated at 35° C. for 16 h. Severaldrops of chloroform were added to prevent the growth of adventitiouscontaminants during incubation. Samples were removed before and afterincubation to measure reducing sugars by the DNS method (see, Wood etal. (1988) Methods in Enzymology 160: 87-112). The degree ofpolymerization (DP) was estimated by dividing the total calculated sugarresidues present in the polymer by the number of reducing ends.

Fermentation Conditions

Fermentations were carried out in 250 ml flasks containing 100 ml ofLuria broth supplemented with 50 g L⁻¹ of carbohydrate. Testcarbohydrates were sterilized separately and added after cooling. Tominimize substrate changes, acid-swollen cellulose, ball-milledcellulose and xylan were not autoclaved. The antibiotic chloramphenicol(200 mg L⁻¹) was added to prevent the growth of contaminating organisms.Flasks were inoculated (10% v/v) with 24-h broth cultures (50 g L⁻¹glucose) and incubated at 35° C. with agitation (100 rpm) for 24-96 h.To monitor cultures, samples were removed daily to determine the ethanolconcentrations by gas chromatography (Dombek et al. (1986) Appl.Environ. Microbiol. 52:975-981).

Methods for Isolating and Identifying a Surrogate Promoter

In order to identify random fragments of Z. mobilis that would serve assurrogate promoters for the expression of heterologous genes inKlebsiella and other host cells, a vector for the efficient cloning ofcandidate promoters was constructed as described in Example 1 (see also,Ingram et al. (1988) Appl. Environ. Microbiol. 54:397-404).

Next, Sau3AI digested Z. mobilis DNA fragments were ligated into theBamHI site of pLO12171 to generate a library of potential promoters.These plasmids were transformed into E. coli DH5α for initial screening.Of the 18,000 colonies individually tested on CMC plates, 75 clonesproduced larger yellow zones than the control (pLOI2173). Plasmids fromthese 75 clones were then transformed into K. oxytoca M5A1, re-tested,and found to express high levels of celZ in this second host.

Recombinant Klebsiella Hosts for Producing Polysaccharase

The high expressing clones (pLOI2177 to pLOI2194) with the largest zoneson CMC plates indicating celZ expression were grown in LB broth andassayed for glucanase activity (Table 5). TABLE 5 Evaluation ofpromoters for celZ expression and secretion in K. oxytoca M5A1 Secretiongenes No secretion genes present (pCPP2006) Secreted Total activityactivity Total activity Secreted activity Plasmids^(a) (IU L⁻¹)^(b) (IUL⁻¹) (IU L⁻¹) (IU L⁻¹) PLOI2173 2,450 465 3,190 1,530 PLOI2177 19,7003,150 32,500 13,300 PLOI2178 15,500 2,320 21,300 11,500 PLOI2179 15,4002,310 21,400 12,000 PLOI2180 21,400 3,210 30,800 13,600 PLOI2I81 15,6002,490 21,000 11,800 PLOI2182 19,600 3,130 31,100 14,000 PLOI2183 20,7003,320 32,000 14,000 PLOI2184 15,500 2,480 21,200 11,900 PLOI2185 15,1002,420 24,600 11,500 PLOI2186 17,000 2,380 25,700 13,400 PLOI2187 15,8002,210 24,500 12,200 PLOI2188 18,200 2,180 25,600 12,000 PLOI2189 14,8002,360 27,100 12,700 PLOI2190 16,100 2,410 26,500 12,500 PLOI2191 15,8002,210 25,000 12,400 PLOI2192 15,100 1,810 24,900 12,500 PLOI2193 16,7002,010 24,600 12,800 PLOI2194 15,400 2,770 21,500 11,900^(a)pLOI2173 contains the celZ gene with the original promoter, allothers contain the celZ gene with a Z. mobilis DNA fragment which servesas a surrogate promoter.^(b)Glucanase (CMCase) activities were determined after 16 h of growthat 30° C.

Activities with these plasmids were up to 8-fold higher than with thecontrol plasmid containing the native celZ promoter (pLOI2173). The fourplasmids which produced the largest zones (pLOI2177, pLO12180, pLOI2182and pLO12183) also produced the highest total glucanase activities(approximately 30,000 IU L⁻¹). One of these plasmids, pLOI2183, wasselected for chromosomal integration.

Chromosomal Integration of a Polysaccharase Gene

To stably incorporate a desirable polysaccharase gene into a suitablehost cell, e.g., Klebsiella P2 strain, a novel vector (pLOI2306) wasconstructed to facilitate the isolation of a DNA fragment which lackedall replication functions but contained the celZ gene with surrogatepromoter, a selectable marker, and a homologous DNA fragment forintegration (FIG. 7). Two AscI sites were added to pUC19 by inserting alinker (GGCGCGCC; SEQ ID NO: 11) into Klenow-treated NdeI and SapI siteswhich flank the polylinker region to produce pLOI2302. A blunt fragmentcontaining the tet resistance marker gene from pBR322 (excised withEcoRI and Aval, followed by Klenow treatment) was cloned into the PstIsite of pLOI2302 (cut with PstI, followed by Klenow treatment) toproduce pLOI2303. To this plasmid was ligated a blunt fragment of K.oxytoca M5A1 chromosomal DNA (cut with EcoRI and made blunt with Klenowtreatment) into the SmaI site of pLOI2303 to produce (pLOI2305). TheEcoRI—SphI fragment (Klenow treated) containing the surrogate Z. mobilispromoter and celZ gene from pLOI2183 was ligated into the EcoRI site ofpLOI2305 (EcoRI, Klenow treatment) to produce pLOI2306. Digestion ofpLOI2306 with AscI produced two fragments, the larger of which containedthe celZ gene with a surrogate promoter, tet gene, and chromosomal DNAfragment for homologous recombination. This larger fragment (10 kbp) waspurified by agarose gel electrophoresis, circularized by self-ligation,and electroporated into the Klebsiella strain P2 and subsequently grownunder selection for tetracycline resistance. The resulting 21tetracycline-resistant colonies were purified and tested on CMC platesfor glucanase activity. All were positive with large zones indicatingfunctional expression of the celZ gene product.

Clones used to produce the recombinant strains were tested for thepresence of unwanted plasmids by transforming DH5α with plasmid DNApreparations and by gel electrophoresis. No transformants were obtainedwith 12 clones tested. However, two of these strains were subsequentlyfound to contain large plasmid bands which may contain celZ and thesewere discarded. Both strains with large plasmids contained DNA, whichcould be sequenced with T7 and M13 primers confirming the presence ofmulticopy plasmids. The remaining ten strains contain integrated celZgenes and could not be sequenced with either primer.

The structural features of the novel vector pLOI2306 are schematicallyshown in FIG. 8 and the nucleotide sequence of the vector, includingvarious coding regions (i.e., of the genes celZ, bla, and tet), areindicated in SEQ ID NO: 12 of the sequence listing (the amino acidsequences are disclosed as SEQ ID NOS 22-24). Nucleotide base pairs3282-4281, which represent non-coding sequence downstream of the celZgene (obtained from E. chrysanthemi), and base pairs 9476-11544 whichrepresent a portion of the non-coding target sequence obtained from K.oxytoca M5A1, remain to be sequenced using standard techniques (e.g., asdescribed in Sambrook, J. et al., T. Molecular Cloning. A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989); Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons (1992)). Forexample, sufficient flanking sequence on either side of theaforementioned unsequenced regions of the pLOI2306 plasmid is providedsuch that sequencing primers that correspond to these known sequencescan be synthesized and used to carry out standard sequencing reactionsusing the pLOI2306 plasmid as a template.

Alternatively, it will be understood by the skilled artisan that theseunsequenced regions can also be determined even in the absence of thepLOI2306 plasmid for use as a template. For example, the remaining celZsequence can be determined by using the sequence provided herein (e.g.,nucleotides 1452-2735 of SEQ ID NO: 12) for synthesizing probes andprimers for, respectively, isolating a celZ containing clone from alibrary comprising E. chrysanthemi sequences and sequencing the isolatedclone using a standard DNA sequencing reaction. Similarly, the remainingtarget sequence can be determined by using the sequence provided herein(e.g., nucleotides 8426-9475 of SEQ ID NO: 12) for synthesizing probesand primers for, respectively, isolating a clone containing targetsequence from a library comprising K. oxytoca M5A1 EcoRI fragments(e.g., of the appropriate size) and sequencing the isolated clone usinga standard DNA sequencing reaction (a source of K. oxytoca M5A1 wouldbe, e.g., ATCC 68564 cured free of any plasmid using standardtechniques). The skilled artisan will further recognize that the makingof libraries representative of the cDNA or genomic sequences of abacterium and the isolation of a desired nucleic acid fragment from sucha library (e.g., a cDNA or genomic library), are well known in the artand are typically carried out using, e.g., hybridization techniques orthe polymerase chain reaction (PCR) and all of these techniques arestandard in the art (see, e.g., Sambrook, J. et al., T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989);Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); and PCRHandbook Current Protocols in Nucleic Acid Chemistry, Beaucage, Ed. JohnWiley & Sons (1999) (Editor)).

Heterologous Gene Expression Using a Surrogate Promoter and Integratedor Plasmid-Based Constructs

The ten integrated strains (SZ1-SZ10) were investigated for glucanaseproduction in LB sorbitol broth (Table 6). All produced 5,000-7,000IUL⁻¹ of active enzyme. Although this represents twice the activityexpressed from plasmid pLOI2173 containing the native celZ promoter, theintegrated strains produced only ⅓ the glucanase activity achieved by P2(pLO12183) containing the same surrogate Z. mobilis promoter (Table 5).The reduction in glucanase expression upon integration may be attributedto a decrease in copy number (i.e., multiple copy plasmid versus asingle integrated copy).

Secretion of Glucanase EGZ

K. oxytoca contains a native Type II secretion system for pullulanasesecretion (Pugsley (1993) Microbiol. Rev. 57:50-108), analogous to thesecretion system encoded by the out genes in Erwinia chrysanthemi whichsecrete pectate lyases and glucanase (EGZ) (Barras et al. (1994) Annu.Rev. Phytopathol. 32:201-234; He et al. (1991) Proc. Natl. Acad. Sci.USA. 88: 1079-1083). Type II secretion systems are typically veryspecific and function poorly with heterologous proteins (He et al.(1991) Proc. Natl. Acad. Sci. USA. 88: 1079-1083; Py et al. (1991) FEMSMicrobiol. Lett. 79:315-322; Sauvonnet et al. (1996) Mol. Microbiol. 22:1-7). Thus as expected, recombinant celZ was expressed primarily as acell associated product with either M5A1 (Table 5) or P2 (Table 6) asthe host. About ¼ (12-26%) of the total recombinant EGZ activity wasrecovered in the broth. With E. coli DH5α, about 8-12% of the totalextracellular EGZ was present. Thus the native secretion system in K.oxytoca may facilitate partial secretion of recombinant EGZ.

To further improve secretion of the desired products, type II secretiongenes (out genes) from E. chrysanthemi EC16 were introduced (e.g., usingpCPP2006) to facilitate secretion of the recombinant EGZ from strainP86021 in ethanologenic strains of K. oxytoca (Table 5 and Table 6). Formost strains containing plasmids with ceIZ, addition of the out genesresulted in a 5-fold increase in extracellular EGZ and a 2-fold increasein total glucanase activity. For strains with integrated celZ, additionof the out genes resulted in a 10-fold increase in extracellular EGZ anda 4-fold increase in total glucanase activity. In both cases, the outgenes facilitated secretion of approximately half the total glucanaseactivity. The increase in EGZ activity resulting from addition of theout genes may reflect improved folding of the secreted product in bothplasmid and integrated celZ constructs. The smaller increase observedwith the pUC-based derivatives may result from plasmid burden andcompetition for export machinery during the production of periplasmicβ-lactamase from the bla gene on this high copy plasmid.

Two criteria were used to identify the best integrated strains of P2,growth on solid medium containing high levels of chloramphenicol (amarker for high level expression of the upstream pdc and adhB genes) andeffective secretion of glucanase with the out genes. Two recombinantstrains were selected for further study, SZ2 and SZ6. Both produced24,000 IU L⁻¹ of glucanase activity, equivalent to approximately 5% ofthe total cellular protein (Py et al. (1991) Protein Engin. 4:325-333).

Substrate Depolymerization

The substrate depolymerization of the recombinant EGZ was determined tobe excellent when applied to a CMC source (Table 7). When applied toacid swollen cellulose, the activity of the glucanase was less than 10%of the activity measured for CMC activity. Little activity was notedwhen the polysaccharase was applied to Avicel® or xylan. However, whenallowed to digest overnight, the EGZ polysaccharase resulted in ameasurable reduction in average polymer length for all substrates. CMCand acid-swollen cellulose were depolymerized to an average length of 7sugar residues. These cellulose polymers of 7 residues are marginallysoluble and, ideally, may be further digested for efficientmetabolization (Wood et al. (1992) Appl. Environ. Microbiol.58:2103-2110). The average chain length of ball-milled cellulose andAvicel® was reduced to ⅓ of the original length while less than a singlecut was observed per xylan polymer. TABLE 6 Comparison of culturegrowth, glucanase production, and secretion from ethanologenic K.oxytoca strains containing integrated celZ Growth Glucanase productionand secretion (IU U) on solid Adding secretion system medium Nosecretion system (pCPP2006) (600 mg L⁻ Total Secreted Secreted Strains¹CM) activity activity Total activity activity P2 ++++ 0 0 0 0 SZ1 ++6,140 1,600 26,100 14,300 SZ2 ++++ 6,460 1,160 23,700 11,400 SZ3 +++5,260 1,320 18,400 8,440 SZ4 +++ 7,120 1,070 23,200 9,990 SZ5 + 6,0001,080 29,300 15,500 SZ6 ++++ 7,620 1,520 24,300 11,900 SZ7 + 6,650 1,33028,800 15,500 SZ8 +++ 7,120 854 28,700 14,900 SZ9 ++ 7,530 1,130 26,70012,800 SZ10 +++ 4,940 939 17,000 6,600Glucanase (CMCase) activities were determined after 16 h of growth at30° C.

TABLE 7 Depolymerization of various substrates by EGZ from cell freebroth of strain SZ6 (pCPP2006) Estimated degree of polymerization EnzymeAfter Substrates activity (IU/L) Before digestion digestionCarboxymethyl cellulose 13,175 224 7 Acid-Swollen cellulose 893 87 7Ball-milled cellulose 200 97 28 Avicel ® 41 104 35 Xylan from oat spelts157 110 78Strain SZ6 (pCPP2006) was grown in LB-sorbitol broth for 16 h as asource of secreted EGZ.Saccharifcation and Fermentation Ability of a Biocatalyst

To be useful, addition of celZ and out genes to strain P2 must notreduce the fermentative ability of the resulting biocatalyst. Acomparison was made using glucose and cellobiose (Table 8). All strainswere equivalent in their ability to ferment these sugars indicating alack of detrimental effects from the integration of celZ or addition ofpCPP2006. These strains were also examined for their ability to convertacid-swollen cellulose directly into ethanol. The most active constructSZ6 (pCPP2006) produced a small amount of ethanol (3.9 g L⁻¹) fromamorphous cellulose. Approximately 1.5 g L⁻¹ ethanol was presentinitially at the time of inoculation for all strains. This decreasedwith time to zero for all strains except SZ6 (pCPP2006). Thus theproduction of 3.9 g L⁻¹ ethanol observed with SZ6 (pCPP2006) mayrepresent an underestimate of total ethanol production. However, atbest, this represents conversion of only a fraction of the polymerpresent. It is likely that low levels of glucose, cellobiose, andcellotriose were produced by EGZ hydrolysis of acid swollen celluloseand fermented. These compounds can be metabolized by the nativephosphoenolpyruvate-dependent phosphotransferase system in K. oxytoca(Ohta K et al., (1991) Appl. Environ. Microbiol. 57:893-900; Wood et al.(1992) Appl. Environ. Microbiol. 58:2103-2110). TABLE 8 Ethanolproduction by strain SZ6 containing out genes (pCPP2006) and integratedcelZ using various substrates (50 g L⁻¹) Ethanol production (g L⁻¹)Strains Glucose Cellobiose Acid-swollen cellulose P2 22.9 22.7 0 P2(pCPP2006) 22.6 21.3 0 SZ6 21.5 19.7 0 SZ6 (pCPP2006) 22.7 21.2 3.9Initial ethanol concentrations at the time of inoculation wereapproximately 1.5 g L⁻¹ for all cultures. With acid swollen cellulose asa substrate, these levels declined to 0 after 72 h of incubation for allstrains except SZ6 (pCP206).

EXAMPLE 3 Synergistic Hydrolysis of Carboxymethyl Cellulose andAcid-Swollen Cellulose by Two Endoglucanases (EGZ and EGY) from Erwiniachrysanthemi

This example describes production of the endoglucanases EGY and EGZ byrecombinant E. coli and the synergistic hydrolysis of carboxymethylcellulose (CMC) and acid-swollen cellulose by these endoglucanases.

Throughout this example, the following materials and methods are usedunless otherwise stated.

Materials and Methods

Bacteria, Plasmids and Culture Conditions

Bacterial strains and plasmids used in this study are listed in Table 9.TABLE 9 Strains and plasmids used Strains / Plasmids DescriptionsReferences / Sources Strains Escherichia coli DH5α lacZ M15 recABethesda Research Laboratory B Prototrophic ATCC11303 HB101 recA lacYrecA ATCC37159 TOP10F' This strain expresses the lac repressorInvitrogen (lacI^(q) gene) from an F episome Plasmids pCR2.1-TOPO TOPO ™TA Cloning vector, Ap^(r), Km^(r) Invitrogen pRK2013 Km^(r) mobilizinghelper plasmid (mob⁺) ATCC pCPP2006 Sp^(r), ca. 40 kbp plasmid carryingthe He, et al. (1991) Proc. complete out genes from E. chrysanthemiNatl. Acad Sci. USA EC16 88:1079-1083. pLOI1620 Ap^(r), celZ gene andits native promoter Beall, et al. (1993) J. from E. chrysanthemi P86021Indust. Microbiol. 11:151-155. pMH18 Ap^(r), celY gene and its nativepromoter Guiseppi, et al (1991) from E. chrysanthemi 3937 Gene106:109-114. pLOI2311 celY gene (without native promoter), See textcloned into pCR2.1-TOPO vector and oriented for expression from the lacpromoterEscherichia coli DH5α and TOPO10OF′ were used as hosts for plasmidconstructions. The celZ gene was cloned from E. chrysanthemi P86021(Beall, et al. (1993) J. Indust. Microbiol. 11:151-155). The celY genewas cloned by Guiseppi et al. ((1991) Gene 106:109-114) from E.chrysanthemi 3937. The out genes were cloned by He et al. ((1991) Proc.Acad Sci. USA 88:1079-1083) from E. chrysanthemi EC16.

E. coli cultures were grown at 37° C. in Luria-Bertani broth (LB)containing per liter: 10 g Difco tryptone, 5 g Difco® yeast extract, and5 g sodium chloride or on solid LB medium containing agar (1.5%). Cloneswere screened for endoglucanase production using the Congo Red method(Wood, et al. (1989) Biochem. J. 260:37-43). Indicator plates wereprepared by supplementing LB agar with low viscosity CMC (0.3%).Ampicillin (50 μg/ml), kanamycin (50 μg/ml) and spectinomycin (100μg/ml) were added as appropriate for selection.

Genetic Methods

Standard methods were used for plasmid construction and analyses(Ausubel, et al. (1987) Current Protocols in Molecular Biology. NewYork: John Wiley and Sons, Inc). The coding region for celY wasamplified by the polymerase chain reaction using pMH18 as the templatewith the following primer pairs: N-terminus 5′CTGTTCCGTTACCAACAC3 (SEQID NO:13)′, C-terminus 5′GTGAATGGGATCACGAGT3′ (SEQ ID NO:14). The E.chrysanthemi out genes (pCPP2006) were transferred by conjugation usingpRK2013 for mobilization (Zhou, et al. (1999) B. Appl. Environ.Microbiol. 65:2439-2445). DNA was sequenced by the dideoxy method usinga LI-COR Model 4000-L DNA sequencer and fluorescent primers.

Enzyme Assay

Endoglucanase activity was determined in vitro using CMC as a substrate.Appropriate dilutions of cell-free culture broth (extracellularactivity) or broth containing cells that had been disrupted byultrasound (total activity) were assayed at 35° C. in 50 mM citratebuffer (pH 5.2) containing low viscosity CMC (20 g per liter). Reactionswere terminated by heating in a boiling water bath for 10 min. Reducingsugars were measured using 3,5-dinitrosalicylic acid reagent withglucose as a standard (Wood, et al. (1988) Methods Enzymology160:87-112). Enzyme activity (CMCase) is expressed as lmol reducingsugar released per min (IU). Results are an average of two or moredeterminations.

Synergism

Stationary phase cultures of DH5α(pLOI1620+pCPP2006) and DH5α(pLOI2311)were sonicated and centrifuged as described in Zhou, et al. (1999),supra, as a source of EGZ and EGY, respectively. These were diluted asnecessary to provide equal CMCase activities. Mixtures of EGZ and EGYwere tested for synergy at 35° C. in 50 mM citrate buffer (pH5.2)containing CMC (20 g/L) or acid swollen cellulose (20 g per liter). Fortests with Avicel® (20 g per liter), enzyme preparations were mixedwithout prior dilution. Hydrolyzed samples of acid-swollen cellulose andAvicel® were centrifuged (10,000×g, 5 min) to remove insoluble materialprior to the determination of reducing sugars.

The effect of sequential additions of EGZ and EGY was also investigated.Substrates were hydrolyzed with a single enzyme for 4 hours and theninactivated by boiling for 20 minutes. After cooling, the second enzymewas added and incubated for an additional 4 hours. Control experimentswere conducted with both enzymes together (4 hours) and with each enzymealone (4 hours). Samples were analyzed for reducing sugar. In somecases, products were also analyzed by thin layer chromatography.

The degree of synergism for enzyme mixtures was calculated as theobserved activity divided by the sum of predicted contributions from EGYalone and EGZ alone (Riedel, et al. (1997) FEMS Microbiol. Lett.147:239-243).

Hydrolysis of Cellooligosaccharides

Hydrolysis products from cellobiose, cellotriose, cellotetraose,cellopentaose, acid-swollen cellulose (Wood, et al. (1988), supra) andAvicel® were analyzed by thin layer chromatography. For these analyses,15 μl of 1% substrate was mixed with 45 μl of crude enzyme (0.07 IU),incubated at 35° C. for 2 hours and terminated by heating in a boilingwater bath. Hydrolysates were spotted on Whatman 250 μm Silica-gel 150Aplates and developed for approximately 4 hours using the solvent systemdescribed by Kim, (1995) Appl. Environ. Microbiol. 61:959-965). Byvolume, this solvent contained 6 parts chloroform, 7 parts acetic acid,and 1 part water. Sugars were visualized by spraying of 6.5 mMN-(1-naphthyl) ethylenediamine dihydrochloride and heating at 100° C.for approximately 10 min (Bounias, (1980) Anal. Biochem. 106:291-295).

Materials and Chemicals

Tryptone and yeast extract were products of Difco (Detroit, Mich.).Antibiotics, low viscosity CMC, cellobiose, cellotriose, andcellotetraose were obtained from the Sigma Chemical Company (St. Louis,Mo.). Cellopentaose was obtained from V-Lab (Covington, La.). Avicel®was purchased from Fluka Chemika (Buchs, Switzerland).

Production of EGY and EGZ by Recombinant E. coli

Low levels of EGY activity were produced by native E. chrysanthemi 3937and by recombinant E. coli harboring plasmid pMH18 (Boyer, et al.((1987) Eur. J. Biochem. 162:311-316; Guiseppi, et al., supra). Poorexpression from the high copy plasmid in E. coli was attributed topromoter function and a putative requirement for a celY activatorprotein (Guiseppi, et al., supra). A new clone was constructed toproduce higher levels of EGY for our investigations of synergy. The EGYcoding region (without promoter) was amplified using the polymerasechain reaction and cloned behind the lac promoter in pCR2.1-TOPO. Theresulting plasmid, pLOI2311, was strongly positive on CMCase indicatorplates. Replacement of the native promoter with the lac promoterincreased celY expression by approximately 10-fold, from 165 IU/L to1800 IU/L (See Table 10, below). TABLE 10 Effect of E. chrysanthemi outgenes on the expression and secretion of celY and celZ in E. coli DH5αout genes present (pCCP2006) No out genes Total Extracellular TotalApparent Extracellular CMCase Apparent Enzyme Growth CMCase^(a) CMCasesecretion CMCase^(a) (IU per secretion expressed Promoter (hr) (IU perliter) (IU per liter) (%) (IU per liter) liter) (%) EGY Native promoter24 136 165 82 136 180 76 (pMH18) lac promoter 8 208 266 78  nd^(b) Nd nd(pLOI2311) 16 1,420 1,590 90 nd Nd nd 24 1,650 1,800 90 1,360 1,510 90EGZ Native plus 8 130 1,320 10 6,710 7,4600 90 EGZ lac promoter 16 1,2009,030 13 13,400 19,700 68 (pLOI1620) 24 1,800 12,500 14 23,600 36,800 64^(a) Secreted or released CMCase activity in the culture supernatant.^(b) Abbreviation: nd, not determined.

Approximately 90% of EGY activity was found in the extracellular milieu.Expression of celZ was included for comparison (See Table 10). Highlevels of EGZ were produced by E. coli harboring plasmid pLOI1620.Extracellular EGZ and total EGZ activity were further increased byaddition of the E. chrysanthemi out genes (pCPP2006) as reportedpreviously (Zhou, et al. (1999) Appl. Environ. Microbiol. 65:2439-2445).Unlike EGZ, however, EGY activity was not affected by the presence ofout genes. Maximal EGY and EGZ activities were obtained from 24-hourcultures. The supernatants from disrupted cultures of DH5α containingpLOI23 11 or pLOI 1620 and pCPP2006 (out genes) were used as a source ofEGY and EGZ, respectively, for further investigations.

Synergistic Action of EGY and EGZ with CMC as a Substrate

Initial studies examining the combined actions of EGY and EGZ wereconducted with CMC (20 g per liter) for a single incubation time (FIG.9). Disrupted cell preparations containing EGY and EGZ were each dilutedto equal activities (CMCase) and combined in different proportions tomaintain a constant sum of individual activities. EGY and EGZ weretested individually as controls. All mixtures of EGY and EGZ weresignificantly higher than either enzyme assayed alone indicating asynergistic interaction. The synergistic effect increased with theproportion of EGZ. Maximal synergy (1.42) was observed with a ratio ofEGZ to EGY activities of 9 to 1 and 19 to 1.

Further experiments examined the effect of incubation time using CMC asthe substrate and an activity ratio of 9 to 1 for EGZ and EGY,respectively (FIG. 9). EGZ and EGY alone were included as controls. Thesynergistic effect of combining EGZ and EGY was clearly evident as anincrease in the rate and extent of hydrolysis. Calculated synergyincreased with incubation time. At the end of the incubation (4 hours),the concentration of reducing sugars was 1.8-fold higher in the mixedenzyme preparation than predicted by the arithmetic sum of individualEGZ and EGY activities, i.e., synergistic activity.

Effect of Substrate (CMC) Concentration on Synergy

The effect of substrate concentration on the synergy between EGZ and EGYwas also studied (See Table 11, below). Increasing the CMC concentrationfrom 2.5 g per liter to 20 g per liter increased the observed synergyfrom 1.12 to 1.89. Based on the specific activities of EGZ and EGY and amaximal synergism of 1.89, the enzyme turnover rate for the combinationwas 8-fold that of purified EGY alone, and 1.5-fold that of purified EGZalone. TABLE 11 Effect of substrate concentration on synergy Reducingsugar released (μmole/ml)^(a) CMC Substrate EGZ (9) + (g/L) EGZ (10)^(b)EGY (10)^(b) EGY (1)^(b) Synergy^(a,c) 20 3.98 ± 0.04 3.83 ± 0.04 7.51 ±0.07 1.89 ± 0.02 10 4.53 ± 0.01 2.91 ± 0.07 5.38 ± 0.04 1.25 ± 0.01 5.02.87 ± 0.01 1.18 ± 0.04 2.92 ± 0.04 1.08 ± 0.02 2.5 1.42 ± 0.01 0.50 ±0.04 1.49 ± 0.01 1.12 ± 0.01^(a)Average ± standard deviation.^(b)EGZ and EGY were diluted to equal CMCase activities. Reactions (0.15IU/ml) contained 9 parts of EGZ and 1 part of EGY. As controls, EGZ(0.15 IU/ml) and EGY (0.15 IU/ml) were each tested individually.^(c)Synergy was calculated as the observed activity divided by the sumof predicted contributions from EGY alone (10%) plus EGZ alone (90%).

EGY was more sensitive to substrate concentration than EGZ. Increasingthe CMC concentration resulted in an 8-fold increase in reducing sugarproducts with EGY but only a 3-fold increase with EGZ. Based on a doublereciprocal plot of the data in Table 11, apparent Km values of 104, 12,and 38 g per liter were estimated for EGY, EGZ and the combination ofboth enzymes (9 parts EGZ+1 part EGY), respectively. The higher apparentKm for EGY is consistent with a requirement for longer substratemolecules.

The extent of CMC hydrolysis was also examined by determining theapproximate size of hydrolysis products. CMC (1.25 g per liter) wasincubated (4 hours, 0.75 IU CMCase/ml) with EGY, EGZ, and a combinationof both enzymes (9 parts EGZ plus 1 part EGY). Chain length wasestimated based on the reducing sugar assay before (250 glucosyl units)and after incubation. The average chain length was substantially reducedby all three enzyme preparations. EGZ was more effective in reducingchain length than EGY, 3.6 versus 10.7 glucosyl residues, respectively.The combination of both enzymes resulted in a synergistic action.Simultaneous hydrolysis with both enzymes reduced the average size ofthe hydrolysis products to 2.3 glucosyl residues, 36% lower than EGZalone and 79% lower than EGY alone. These results confirm that EGZreadily hydrolyzes both large CMC polymers and smaller saccharides. Theaction of EGY appears more limited, primarily hydrolyzing large polymerswith greater than 10 glucosyl units.

Sequential and Simultaneous Hydrolysis of CMC with EGZ and EGY

The mechanism of synergistic action between EGZ and EGY was furtherinvestigated by comparing the effects of sequential hydrolysis withindividual endoglucanases to that of simultaneous hydrolysis by amixture of both enzymes (See Table 12, below). Again synergy wasobserved for the simultaneous actions of both enzymes. No synergy wasobserved for the sequential hydrolysis of CMC when EGZ was used as thefirst enzyme, followed by digestion with EGY (after heat-inactivation ofEGZ). In contrast, full synergy was retained when CMC was first treatedwith EGY, followed by EGZ (after heat-inactivation of EGY). Theseresults indicate that synergy can be achieved by the independentactivities of EGY and EGZ. Enzymatic modification of the substrate byEGY increased the rate and extent of subsequent hydrolysis by EGZ. Theseresults provide further evidence that EGY and EGZ function quitedifferently. EGY appears to primarily reduce the chain length of largepolymers while EGZ appears to act more randomly, hydrolyzing both largeand small substrate molecules. TABLE 12 Sequential and simultaneoushydrolysis of CMC by EGZ and EGY Predicted activity from the arithmeticsum of EGY and Measured reducing sugar EGZ Enzyme (relativeproportion)^(a) released (μmole/ml)^(b) (μmole/ml)^(c) Synergy^(b,d) EGZ(10) + EGY (0) 4.65 ± 0.08 4.65 1.00 ± 0.02 EGZ (0) + EGY (10) 4.14 ±0.04 4.14 1.00 ± 0.01 EGZ (9) + EGY (1) 8.28 ± 0.08 4.60 1.80 ± 0.02(simultaneously) EGZ (9), then EGY (1). (sequential) 4.86 ± 0.23 4.601.06 ± 0.05 EGY (1), then EGZ (9). (sequential) 8.75 ± 0.14 4.60 1.90 ±0.03^(a)EGZ and EGY were diluted to equal CMCase activities. Bothsimultaneous and sequential hydrolysis reactions (0.15 IU/ml) wereinvestigated using 9 parts of EGZ and 1 part of EGY. In the sequentialhydrolysis experiments, the first enzyme was incubated with substratefor 4 hours and inactivated by boiling for 20 min. After cooling, thesecond enzyme was added and incubated for an additional 4 hours. Allreactions were terminated by boiling.^(b)Average ± standard deviation (3 experiments).^(c)Calculated sum of individual EGY and EGZ activities.^(d)Synergy was calculated as the observed activity divided by the sumof predicted contributions from EGY alone (10%) plus EGZ alone (90%).Synergistic Action on Acid-Swollen and Crystalline Cellulose

Potential synergy was investigated using acid-swollen cellulose as thesubstrate and a 9 to 1 ratio of EGZ:EGY based on CMCase activities (FIG.9). Since the activities of EGZ and EGY with acid-swollen cellulose arelower than those with CMC (Boyer, et al. (1987) Eur. J. Biochem.162:311-316), enzyme loading (1.5 IU) and incubation times wereincreased. When assayed individually with acid-swollen cellulose, EGYwas approximately ⅓ as active as EGZ. However, the combination of thesetwo enzymes was significantly more active than the predicted arithmeticsum of individual activities at all time points. The degree of synergywas essentially constant (1.36±0.17) during the 36 hour period ofincubation.

The hydrolysis products from acid-swollen cellulose (6 hours) wereanalyzed by thin layer chromatography (FIG. 10). No soluble saccharideswere observed after incubation with EGY alone. Cellobiose andcellotriose were the primary products from hydrolysis with EGZ alone anda combination of EGY and EGZ. With the combination of both enzymes,higher product levels were evident as darker and larger spots confirminga synergistic action.

The synergistic action of EGZ and EGY was also investigated with Avicel®(FIG. 10), a highly crystalline cellulose. Small amounts of cellobioseand cellotriose were observed as hydrolysis products with EGZ alone andwith the mixture of EGY and EGZ. Due to low activity with Avicel®, highloadings (10 μl) were required on thin layer plates to visualizeproducts. Note that this additional salt increased the relativemigration of oligosaccharide products in comparison to the standards. Nocellooligosaccharide spots were observed with EGY alone. Again synergismwas evident with the combination of EGY and EGZ. Larger and more intensespots were observed corresponding to cellobiose, cellotriose, andcellotetraose with the combined activities than with EGZ alone. The lowactivity with Avicel® as a substrate and the relatively low levels ofproducts are consistent with the hydrolysis of the amorphous rather thanthe crystalline regions of Avicel®. These results indicate that thesynergistic action of EGZ and EGY is not limited to a model substratesuch as CMC. Synergistic hydrolysis was also observed for acid-swollencellulose and the amorphous regions of Avicel®.

Hydrolysis of Cellooligosaccharides

The substrate specificities of EGZ and EGY were further investigatedusing soluble cellooligosaccharides (cellobiose, cellotriose,cellotetraose, and cellopentaose). Hydrolysis products were analyzed bythin layer chromatography (FIG. 11). Cellobiose was not hydrolyzed byEGY, EGZ or a combination of both enzymes. None of thecellooligosaccharides was hydrolyzed by EGY alone (FIG. 11, Panel B). Incontrast, EGZ hydrolyzed cellotetraose and cellopentaose but notcellotriose (FIG. 11, Panel C). EGZ hydrolysis products fromcellotetraose were primarily cellobiose with lesser amounts ofcellotriose and glucose. With cellopentaose as the substrate, EGZproduced approximately equal amounts of cellobiose and cellotrioseindicating a preferential attack on the second or third glycosidic bond.This was further confirmed by examining samples at various times duringthe incubation of cellopentaose with EGZ (FIG. 11, Panel D). Cellobioseand cellotriose progressively accumulated during incubation with acorresponding reduction in cellopentaose. Thus in contrast to therequirement for large substrates by EGY, EGZ hydrolyzes solublecellooligosaccharides containing four or more glucosyl units.

EXAMPLE 4 Integration, Expression, and Extracellular Secretion ofErwinia chrysanthemi Endoglucanase EGY (celY) and EGZ (celY) inEthanologenic Klebsiella oxytoca P2

In this example, the functional integration of both celY and celZ fromE. chrysanthemi into the chromosome of K. oxytoca P2, is described. Alsodescribed is the synergism between recombinant EGY and EGZ and fungalcellulase (Spezyme CE®) during the fermentation of cellulose to ethanolusing simultaneous saccharification and fermentation.

Throughout this example, the following materials and methods are usedunless otherwise stated.

Materials and Methods

Bacteria, Plasmids and Culture Conditions

Strains and plasmids used in this example are listed in Table 13 below.TABLE 13 Strains and Plasmids Strains / plasmids Description Sources /references E. coli strains DH5α lacZ M15 recA Bethesda ResearchLaboratory TOP10F' hsdR mcrA lacZΔM15 endA recA; F' tet lacl InvitrogenHB101 recA lac Y ATCC37159 S17-1 thi pro recA hsdR RP4-2-tet::MuaphA:Tn7 λpir De Lorenzo, et al. (1990) J. Bacteriol. 172:6568-6572. Z.mobilis strain CP4 Prototrophic Ingram, et al. (1999) Biotechnol. Prog.15:855-866. K. oxytoca strains M5A1 Prototrophic Wood, et al. (1992)Appl. Environ. Microbiol. 58:2103- 2110. P2 pfl::pdc adhB cat Wood, etal. (1992) Appl. Environ. Microbiol. 58:2103- 2110. SZ6 pfl::pdc adhBcat; integrated celZ tet SZ12 pfl::pdc adhB cat; integrated celZ celYkan See text SZ21 pfl::pdc adhB cat; integrated celZ celY See text SZ22pfl::pdc adhB cat; integrated celY celZ::aac See text Plasmids pUC18 blacloning vector New England Biolabs pUC19 bla cloning vector New EnglandBiolabs pCR2.1-TOPO TA cloning vector, bla kan Invitrogen pMH18 bla celYfrom E. chrysanthemi 3937 Guiseppi, et al. (1991) Gene 106:109-114.pHPΩ45aaac bla aac source of apramycin gene Blondelet-Rouault, et al.(1997) Gene 190:315-317. pBR322 bla tet cloning vector New EnglandBiolabs pRK2013 kan, mobilizing plasmid ATCC pCPP2006 spm, ca. 40 kbpfragment containing out genes from E. He, et al. (1991) Proc.chrysanthemi EC16 Natl. Acad Sci. USA 88:1079-1083. pFT-A bla low copyvector containing flp recombinase gene Martinez-Morales, et andtemperature conditional pSC101 replicon al. (1999) J. Bacteriol.181:7143-7148. pLOI2224 kan integration vector containing conditionalR6K Martinez-Morales, et replicon and two FRT sites al. (1999) J.Bacteriol. 181:7143-7148. pLOI2307 bla containing celZ gene and asurrogate promoter Zhou, et al. (1999) from Z. mobilis DNA Appl.Environ. Microbiol. 65:2439- 2445. pLOI2311 PCR fragment containing celYgene cloned into Zhou, et al. (2000) J. pCR2.1-TOPO, expressed from lacpromoter Bacteriol. 182:5676- 5682. pLOI2302 pUC19 containing Ascllinkers inserted into blunt NdeI Zhou, etal. (1999) J. and SapI sitesIndust. Microbiol. Biotechnol. 22:600- 607. pLO12316 pUC 18 containingthe celY gene on a Klenow-treated See text EcoRI fragment from pLOI2311inserted into a blunt HincII site, expressed from the lac promoterpLO12317 EcoRI-HindIII fragment from pLO12316 inserted into See text thecorresponding sites of pLOI2302 pLOI2318 Sau3A1 fragment of Z. mobilisDNA fragment See text exhibiting promoter activity inserted into theBamHI site of pLOI2317 pLO12319 Sau3A1 fragment of Z. mobilis DNAexhibiting See text promoter activity inserted into the BamHI site ofpLOI2317 pLOI2320 Sau3A1 fragment of Z. mobilis DNA exhibiting See textpromoter activity inserted into the BamHI site of pLOI2317 pLOI2323Sau3A1 fragment of Z. mobilis DNA exhibiting See text promoter activityinserted into the BamHI site of pLOI2317 pLOI2342 Sau3A1 fragment of Z.mobilis DNA exhibiting See text promoter activity inserted into theBamHI site of pLOI2317 pLOI2348 Random EcoRI fragment of K. oxytoca M5A1DNA See text cloned into EcoRI site of pLOI2323 pLOI2349 EcoRI linkerinserted into the Klenow-treated SphI See text site of pLOI2307 pLOI2350EcoRI fragment (celZ and surrogate promoter) from See text pLOI2349inserted into the EcoRI site of pLOI2224 pLOI2352 AscI fragment (K.oxytoca fragment, Z. mobilis See text promoter fragment and celY) frompLOI2348 inserted into the AscI site of pLOI2350 pLOI2353 EcoRI-AvaIfragment (tet gene) from pBR322 inserted See text into the ClaI site ofpFT-A. pLOI2354 pUC19 derivative in which the multiple cloning sites Seetext from HindIII to SmaI were deleted by digestion, Klenow-treatment,and self-ligation pLOI2355 EcoRI fragment (celZ gene) from pLOI2349inserted See text into the EcoRI site of pLOI2354. pLOI2356 SmaIfragment containing the apramycin resistance See text gene (aac gene)from pHPΩ45aac inserted into the T4 polymerase-treated PstI site ofpLOI2355, disrupting the celZ gene pLOI2357 EcoRI fragment (aac anddisrupted celZ) inserted into See text the EcoRI site of pLOI2224pLOI2358 Subclone of pLOI2323 in which the internal PstI See textfragment was deleted, used for sequencing pLOI2359 Subclone of pLOI2323in which the ClaI-HindIII See text fragment was deleted, used forsequencing

Escherichia coli DH5α and TOPO10F′ were used as hosts during plasmidconstructions. The celZ gene, celY gene, and out genes were cloned asdescribed in Example 3.

E. coli cultures were grown at 37° C. in Luria-Bertani broth (LB)containing per liter: 10 g Difco® tryptone, 5 g Difco 8 yeast extract,and 5 g sodium chloride or on solid LB medium containing agar (1.5%).Sugar was always included in broth (5% glucose or sorbitol) and solidmedia (2% glucose) used for the growth of ethanologenic strains. Cloneswere screened for endoglucanase production using the Congo Red method(Wood et al. (1988) Methods Enzymology 160:87-112). Endoglucanseindicator plates were prepared by supplementing LB agar with 0.3% lowviscosity carboxy methyl cellulose (CMC). Ampicillin (50 μg/ml),apramycin (100 μg/ml), kanamycin (50 μg/ml), chloramphenicol (40 μg/ml)and spectinomycin (100 μg/ml) were used for selection. Ethanologenicstrains of K. oxytoca were maintained at 30° C. on solid LB mediumcontaining glucose (2%) and chloramphenicol (600 μg/ml).

Genetic Methods

Standard methods were used for plasmid construction, analyses, andsequencing. The ribosome-binding site and promoterless coding region ofcelY were amplified by the polymerase chain reaction using pMH18 as thetemplate with the following primer pairs: N-terminus5′CTGTTCCGTTACCAACAC3′ (SEQ ID NO:13), C-terminus 5′GTGAATGGGATCACGAGT3′(SEQ ID NO:14). The E. chrysanthemi out genes (pCPP2006) weretransferred by conjugation using pRK2013 for mobilization. Constructionswere confirmed by sequencing using the dideoxy method and a LI-COR Model4000-L DNA sequencer with fluorescent primers. The E. chrysanthemi celYand celZ genes were introduced into K. oxytoca P2 by electroporationusing a Bio-Rad Gene Pulser®. Recombinants were selected on solid mediumcontaining kanamycin (50 mg/liter) as described in Martinez-Morales, etal. (1999) J. Bacteriology 181:7143-7148, and Zhou, et al. (1999)Indust. Microbiol. Biotechnol. 22:600-607.

Primer Extension Analysis

Promoter regions were identified by mapping the transcriptional startsites using a IRD41-labeled primer fluorescent primers within the codingregions: 5′-ACCATCAGCATCAACGCCCAACAACG-3′ (SEQ ID NO: 15) for celY and5′-GACTGGATGGTTATCCGAATAAGAGAGAGG-3′ (SEQ ID NO: 16) for celZ. Extensionproducts were dissolved in loading buffer and compared to paralleldideoxy sequences using the LI-COR Model 4000-L DNA sequencer (Lincoln,Nebraska).

Enzyme Assay

Endoglucanase activity was determined as described in Example 3.

Fermentation

Simultaneous saccharification and fermentation (SSF) tests wereconducted in unbaffled, 500-ml flasks containing a 200 ml of broth.Flasks were fitted with a rubber stopper and vented with an 18 gaugeneedle. Fermentations were conducted at 35° C. (120 rpm) in LB mediumcontaining 10% Sigmacell 50 (crystalline cellulose). Inocula were grownfor 12 hours in LB containing 5% glucose. Cells were harvested bycentrifugation and resuspended in LB. Each flask was inoculated toprovide an initial density of 16 mg of cells (dry weight).

Materials and Chemicals

Tryptone and yeast extract were products of Difco (Detroit, Michigan).Antibiotics, low viscosity CMC, and Sigmacell 50® were obtained from theSigma Chemical Company (St. Louis, Mo.). The IRD41-labeled fluorescentprimers were purchased from LI-COR, Inc. (Lincoln, Nebr.).

Construction of a Promoter-Probe Vectorfor celY

The celY gene from E. chrysanthemi is poorly expressed from its nativepromoter in E. coli (See Guiseppi, et al (1991) Gene 106:109-114).Accordingly, to increase expression, a promoter-probe vector wasconstructed as follows using celY as the reporter (See FIG. 13). Apromoterless celY coding region with ribosomal-binding site (1.2 kbp)was amplified by PCR using pMH18 as the template and randomly insertedinto the topoisomerase vector, PCR2.1-TOPO. Functional expression ofcelY was confirmed using endoglucanase indicator plates. A cloneoriented to express celY from the lac promoter was selected anddesignated pLOI2311 (5.2 kbp). An EcoRI fragment containing thepromoterless celY gene was isolated from pLOI2311. The ends of thisfragment were blunted using Klenow polymerase prior to ligation into theHincII site of pUC18. A clone oriented to express celY from the lacpromoter was selected (3.9 kbp) and expression confirmed usingendoglucanase indicator plates (pLOI2316). The promoterless celY genewas isolated from pLOI2316 as a 1.2 kbp fragment using EcoRI and HindIIIand inserted into the corresponding sites of pLOI2302 (pUC19 derivative)to reverse the direction of the celY gene. As expected, the resultingconstruct DH5α(pLOI2317) was inactive on endoglucanase indicator platesdue to the lack of a promoter. To facilitate the insertion of DNAfragments containing promoter regions, plasmid pLOI2317 (3.9 kbp)contains a BamHII site in the polylinker region, immediately upstreamfrom the celY gene (See FIG. 13).

Construction of Plasmids with Increased Expression of celY in E. coliDH5α

Sau3A1 fragments of Z. mobilis chromosomal DNA were used to provide aheterologous promoter that would not be subject to native regulatorymechanisms in K. oxytoca or interfere with subsequent integration intothe K. oxytoca chromosome (Zhou, et al. (1999) J. Indust. Microbiol.Biotechnol. 22:600-607).Fragments of 0.5-1.5 kbp were isolated andrandomly ligated into the BamHI site of pLOI2317 to generate a libraryof surrogate promoters (See FIG. 13). Approximately 75,000 colonies werescreened on endoglucanase indicator plates. One-third of the clonesactively produced celY. The most active 100 colonies were identified byzone size, purified, and re-tested. The 30 clones with the largest zonesof activity were grown overnight in LB and assayed for CMCase activity.The five most active are listed in Table 14 below, and exhibitedapproximately 7-fold higher activity than the original clone, pMH18.Plasmid pLOI2323 was selected for further investigation. TABLE 14Expression of celY in DH5α using fragments using Sau3A1 digestionproducts of Z. mobilis chromosomal DNA as surrogate promoters. Plasmidsexpressing Endoglucanase activity (IU/L) celY or celZ ExtracellularTotal % Extracellular pMH18 151 184 82 (native celY promoter) pLOI2317 00 0 (promoterless celY vector) celY expressed from surrogate promoterspLOI2318 1,123 1,257 89 pLOI2319 888 1,023 87 pLOI2320 1,023 1,056 97pLOI2323 1,257 1,291 97 pLOI2342 1,224 1,257 97 pLOI2349 (celZ) 3,41416,234 21All plasmids are pUC derivatives. Endoglucanase activity was measuredusing cultures grown at 37° C. for approximately 16 hours.

The Z. mobilis Sau3A1 fragment (937 bp) in pLOI2323 was sequenced inboth directions (GenBank Accession No. AF305919). Based on a databasecomparison, this fragment appears to be derived from two pieces, a 882bp fragment form Z. mobilis chromosome which corresponds to a previouslysequenced region and a 55 bp fragment from the vector. A BLAST search(National Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/BLAST/) of the translated sequence did notreveal identity to known genes. Four sites of transcriptional initiationwere identified in DH5α (pLOI2323) by primer extension analysisinvolving three different sigma factors, δ³², δ³⁸, and δ⁷⁰ (See FIG.14). Although the differences in intensity were less than 2-fold, thesequence upstream from the most intense start site resembled theconsensus for δ³² (rpoH), the heat shock promoter (Wang, et al. (1 998)J. Bacteriology 180:5626-5631; Wise, et al. (1996) J. Bacteriology178:2785-2793).

Construction of a Vector for the Integration of celY and celZ into theChromosome of K. oxytoca P2

The plasmid pLOI2307 (7.2 kbp) was constructed and used to express celZfrom a surrogate Z. mobilis promoter at high levels in recombinant E.coli DH5α (See Zhou, et al. (1999) B.Appl. Environ. Microbiol.65:2439-2445) and K. oxytoca M5A1 (See Zhou, et al. (1999) Indust.Microbiol. Biotechnol. 22:600-607). To facilitate subcloning of thishybrid celZ gene and promoter (4.5 kbp), an EcoRI linker was insertedinto the T4 polymerase-treated SphI site of pLOI2307 to provide flankingEcoRI sites for convenient excision (pLOI2349). Prior to constructing aplasmid containing celY and celZ, a random 3 kbp fragment ofEcoRI-digested K. oxytoca M5A1 chromosomal DNA was inserted intopLOI2323 containing celY (and surrogate promoter) to serve as a guidefor homologous recombination (pLOI2348; 8 kbp). This 3 kbp M5A1 fragmentwas partially sequenced and appears to encode the complete M5A1 glgPgene. In pLOI2348 (8 kbp), flanking AscI sites allowed the excision of asingle 5.5 kbp fragment containing the M5A1 glgP gene, Z. mobilissurrogate promoter, and E. chrysanthemi celY.

FIG. 15 summarizes the construction of the celY, celZ integration vectorfrom pLOI2349, pLOI2224, and pLOI2348. The recombinant celY and celZgenes containing surrogate promoters and the guide fragment weresequentially inserted into the core integration vector, pLOI2224(Martinez-Moralez, et al., supra) using E. coli S17-1 as the host, toproduce pLOI2352 (12 kbp). The 4.5 kbp EcoRI fragment from pLOI2349containing celZ was inserted into pLOI2224 using an EcoRI site to makepLOI2350 (6.4 kbp). The 5.5 kbp AscI fragment from pLOI2348 containingcelY was inserted into the AscI site of pLOI2350 to make pLOI2352 (12kbp). The fragments containing cel genes were oriented such thatexpression from the surrogate promoters was divergent. The resultingvector contained a R6K replicon that does not function in DH5α or M5A1.The two FRT sites in pLOI2352 facilitate removal of the kanamycin geneand replicon after integration (Martinez-Moralez, et al., supra).

Functional Integration of celY and celZ into the K. oxytoca P2Chromosome

Plasmid pLOI2352 was introduced into P2 by electroporation followed byselection for kanamycin resistance. Approximately 150 colonies wererecovered and all were positive on endoglucanase indicator plates. Tenclones with the largest zones of activity were purified, grown in brothand assayed for endoglucanase activity. These produced 5-6 IU/ml ofendoglucanase activity. One clone was selected for further study anddesignated as SZ12.

Due to the natural resistance of K oxytoca to ampicillin, an additionalantibiotic resistance marker (tet) was added to pFT-A plasmid containingthe flp recombinase to facilitate selection. The tetracycline gene wasisolated as a 1.4 kbp EcoRI to AvaI fragment from pBR322. Aftertreatment with Klenow polymerase, this fragment was ligated into theKlenow-treated ClaI site of pFT-A to produce pLOI2353 (7.0 kbp). Thisplasmid encodes resistance to both ampicillin and tetracycline, the FLPrecombinase (flp) under the control of the tetracycline promoter, and atemperature-conditional pSC101 replicon.

Plasmid pLOI2353 was transformed into SZ12 and plated at 30° C. withselection for tetracyline resistance. The presence of tetracycline alsoinduced flp expression resulting in a deletion of the kanamycin gene andR6K replicon from chromosomally integrated pLOI2353. Of 307tetracycline-resistant colonies tested, >99% retained expression of theendoglucanase genes and were sensitive to kanamycin. Clones werepurified, grown in broth and assayed for endoglucanase activity. Allwere similar and one was designated SZ21(pLOI2353). The helper plasmidwas eliminated from SZ21 by overnight growth at 37° C.

Construction of a celZ Knockout Mutation

To confirm the presence of functional celY in SZ21, a knockout mutationof the chromosomally integrated celZ was constructed by double,homologous recombination using plasmid pLOI2357 (FIG. 16). Plasmid,pUC19 was digested with SmaI and HindIII, treated with Klenowpolymerase, and self-ligated to eliminate many of the polylinker sites(pLOI2354). The remaining EcoRI site was used to insert a 4.5 kbp EcoRIfragment containing the promoter and celZ gene from pLOI2349 to makepLOI2355 (7.2 kbp). The 1.8 kbp SmaI fragment from pHPQ45aac containingthe apramycin resistance gene (aac) was then ligated into the centralregion of celZ, replacing a small internal PstI fragment (after bluntingwith T4 polymerase) to produce pLOI2356 (9.0 kbp). The 6.3 kbp EcoRIfragment from this plasmid was isolated and inserted into the coreintegration vector, pLOI2224, to produce pLOI2357 (8.2 kbp). Thisplasmid contains a conditional R6K replicon and kanamycin resistancegene in addition to a celZ gene that is interrupted by an apramycinresistance gene.

Plasmid pLOI2357 was electroporated into SZ21 with selection forapramycin. Approximately 10% of the recombinants were apramycinresistant and kanamycin sensitive indicating a double homologousrecombination event. These clones exhibited low levels of endoglucanaseproduction on indicator plates (See Table 15, below). One was selectedand designated SZ22. Loss of EGZ and retention of EGY in SZ22 wasconfirmed by SDS-PAGE using the Pharmacia Phast Gel system.

It is interesting to note that cell clumping in liquid culture, typicalof M5A1 and P2, was eliminated by the functional expression of celZ fromintegrated genes or from plasmids. Clumping was not affected by thefunctional expression of celY alone.

Transcriptional Initiation in K. oxytoca SZ21

Primer extension analysis of celY and celZ in SZ21 were similar to thoseobserved in DH5α. A single major transcriptional start was identifiedfor celZ that corresponded precisely to the most prominent start site inDH5α (pLOI2183) which contains the same promoter fragment (Zhou, et al.(1999) J. Indust. Microbiol. Biotechnol. 22:600-607; Zhou, et al. (1999)B. Appl. Environ. Microbiol. 65:2439-2445). DNA immediately upstreamfrom this site resembles the recognition sequence for a σ⁷⁰ promoter(Wang, et al, supra and Wise, et al., supra). As observed with DH5α(pLOI2323) (See FIG. 14), primer extension analysis of celY indicatedthe presence of multiple putative transcriptional starts in SZ21.Although localized in the same regions as the start sites inDH5α(pLOI2323), all bands were of near equal intensities.

Effect of the E. chrysanthemi Out Genes (pCPP2006) on the ExtracellularSecretion of EGY and EGZ in Derivatives of K. oxytoca P2

Table 7 summarizes the endoglucanase activities exhibited bycellulolytic derivatives of ethanologenic K. oxytoca P2. Strain SZ6(Zhou, et al., (1999) J. Indust. Microbiol. Biotechnol. 22:600-607)contains a chromosomally integrated hybrid celZ gene with same promoterfragment used to construct SZ21. Despite the presence of twoendoglucanase genes in SZ21, extracellular and total endoglucanaseactivities were 13% lower in this strain than in SZ6. Most of theendoglucanase activity produced by SZ21 can attributed to celZ. SZ22, acelZ mutant of SZ21, expressed only 11% of the endoglucanase produced bythe parent containing functional celY and celZ genes. In strains SZ6 andSZ21 containing a functional celZ, most of the endoglucanase activity(primarily EGZ) was cell associated. In strain SZ22 containing afunctional celY alone, half of the endoglucanase activity wasextracellular. TABLE 15 Effect of out genes (pCPP2006) on endoglucanaseproduction by derivatives of K. oxytoca P2. CMC zone CMCase Activity(IU/L)^(a) Strains (mm) OD₅₅₀ Extracellular Total Secretion (%) P2 010.5 0 0 0 SZ6 8.5 11.0 1,920 8,800 22 SZ21 6.7 11.0 1,620 7,800 21 SZ222.0 10.0 480 879 55 P2 (pCPP2006) 0 10.0 0 0 0 SZ6 (pCPP2006) 10.8 9.613,800 22,300 62 SZ21 (pCPP2006) 11.5 10.2 20,100 26,900 75 Spezyme CE ®(10 ml/liter)^(b) — — — 27,000 — Spezyme CP ® (10 ml/liter)^(b) — — —33,400 —^(a)Endoglucanase activity was measured using cultures grown at 30° C.in LB containing 5% sorbitol for 24 h.^(b)Dilution equivalent to the highest Spezyme ® level used infermentation experiments (Table 4).

The addition of the out genes (pCPP2006) to recombinant E. coli and K.oxytoca M5A1 harboring celZ can cause a dramatic increase in thefunctional expression of celZ and in the fraction of EGZ secreted intothe extracellular milieu (Zhou, et al., (1999) J. Indust. Microbiol.Biotechnol. 22:600-607; Zhou, et al. (1999) B. Appl. Environ. Microbiol.65:2439-2445). These same effects were observed for ethanologenic K.oxytoca SZ21 containing celZ and celY (See Table 14). Addition of theout genes to SZ22 (inactive celZ) had no effect on the functionalexpression of celY or the extent of EGY secretion. This celZ mutant,SZ22 (pCPP2006), produced only 3% of the total endoglucanase activity(EGY) produced by SZ21 (pCPP2006) containing functional celY and celZgenes. The secreted endoglucanase produced by SZ21 with the out geneswas substantially higher than the sum of the individual activitiesexpressed from the same respective promoters in SZ6 (EGZ) and SZ22(EGY), consistent with synergism between these two enzymes. In thisassay, synergy is estimated to be 1.4-fold the arithmetic sum of theindividual activities (SZ6(pCPP2006) and SZ22(pCPP2006)) for thecombination of extracellular enzymes produced by SZ21(pCPP2006).

Synergism Between Recombinant E. chrysanthemi Endoglucanase (EGZ andEGY) and Fungal Cellulase (Spezyme CE®) During the Fermentation ofCellulose to Ethanol

SSF tests in flasks were used without pH control to evaluate thecombined effects of fungal cellulase (Spezyme®) and cellulase enzymesproduced by the biocatalysts on ethanol production from Sigmacell 50®, ahighly crystalline substrate. Results are indicated in Table 16, below.TABLE 16 Maximum ethanol production Genencor Spezyme ® Fermentation^(a))Addition Ethanol ± SD % Control ± Strain Type (ml/liter) N(g/liter)^(b)) SD P2(pCPP2006) none 0 3 0.23 ± 0.01   100 ± 2.0SZ6(pCPP2006) none 0 3 0.28 ± 0.02 *   124 ± 8.5 SZ21(pCPP2006) none 0 30.26 ± 0.02 *   116 ± 8.5 SZ22(pCPP2006) none 0 3 0.24 ± 0.01   107 ±1.0 P2(pCPP2006) CE 5.0 6 13.7 ± 0.3   100 ± 2.0 SZ6(pCPP2006) CE 5.0 613.8 ± 0.3   101 ± 2.4 SZ21(pCPP2006) CE 5.0 6 16.0 ± 0.5 **   117 ± 3.5SZ22(pCPP2006) CE 5.0 6 15.2 ± 0.3 **   112 ± 1.5 P2(pCPP2006) CE 10.0 620.7 ± 0.5   100 ± 2.1 SZ6(pCPP2006) CE 10.0 6 21.2 ± 0.1 103.4 ± 0.4SZ21(pCPP2006) CE 10.0 6 24.6 ± 0.5 **   121 ± 2.3 SZ22(pCPP2006) CE10.0 6 25.2 ± 1.1 **   122 ± 5.0 P2(pCPP2006) CP 5.0 6 15.2 ± 0.3   100± 1.7 SZ21(pCPP2006) CP 5.0 6 17.8 ± 0.4**   116 ± 2.2 P2(pCPP2006) CP10.0 6 25.3 ± 0.7   100 ± 2.6 SZ21(pCPP2006) CP 10.0 6 27.2 ± 0.3 **  107 ± 1.2^(a)) Cultures without added cellulose (100 g/liter) orSpezyme ®produced 0.22 ± 0.01 g/liter of ethanol. Spezyme ®containedapproximately 100 FPU/ml. Addition of 5 ml and 10 ml ofSpezyme ®corresponds to 5 FPU/g and 10 FPU/g cellulose, respectively.^(b)) Student t-test shows that there is significant difference inethanol production compared to the respective P2 controls at eachSpezyme ®dilution. P value ≦ 0.001 is indicated by a two asterisks.P value ≦ 0.05 is indicated by a single asterisk.

Although very low levels of ethanol were produced by all strains in theabsence of Spezyme®, strains SZ6(pCPP2006) and SZ21 (pCPP2006)containing functional celZ genes produced higher levels of ethanol(p<0.05) than strain SZ22(pCPP2006) containing only a functional celYgene and strain P2(pCPP2006) lacking endoglucanase genes. In the absenceof both Spezyme® and Sigmacell 50®, all strains produced 0.22 g/Lethanol. The additional increment of ethanol produced by SZ6(pCPP2006)and SZ21 (pCPP2006) during incubation with Sigmacell 50 is attributed tohydrolysis of the small fraction of amorphous cellulose in the substrateby EGZ (Zhou, et al. (1999) J. of Industrial Microbiol. Biotechnol.22:600-607; Zhou, et al. (1999) B. Appl. Environ. Microbiol.65:2439-2445; Zhou, S., et al. (2000) J. Bacteriol. 182:5676-5682).Digestion of amorphous cellulose by EGY produces saccharides that aretoo large to be transported and metabolized without further hydrolysis(Zhou, S., et al. (2000) J. Bacteriol. 182:5676-5682).

Spezyme CE® and Spezyme CP® contain a commercially optimized combinationof endoglucanase, exoglucanase, and cellobiase activities (Beguin, etal. (1994) FEMS Microbiol. Rev. 13:25-58; Boyer, et al. (1987) Eur. J.Biochem. 162:311-316; Nieves, et al. (1998) World Journal ofMicrobiology and Biotechnology 14:310-314; Ohmiya, et al. (1997)Biotechnol. Genetic Eng. Rev. 14:365-414). Despite this optimization,Spezyme®-supplemented fermentations with two of the endoglucanaseproducing biocatalysts, SZ21(pCPP2006) and SZ22(pCPP2006), producedsignificantly higher levels of ethanol than the control P2(pCPP2006)which lacks endoglucanase genes. The combinations of Spezyme® andSZ21(pCPP2006) and SZ22(pCPP2006) were synergistic in terms of ethanolproduction, up to 20% higher than the sum of ethanol produced by eachindividually (p≦0.005). Synergy was observed for both dilutions ofSpezyme CE®, and for Spezyme CP®. This synergistic effect can beattributed primarily to EGY since this is the only endoglucanaseproduced by SZ22(pCPP2006). No synergy was observed for SZ6(pCPP2006)which produces only EGZ.

EXAMPLE 5 Simultaneous Saccharification and Fermentation of AmorphousCellulose to Ethanol by Recombinant Klebsiella oxytoca SZ21 WithoutSupplemental Cellulase

In this example, a derivative of Klebsiella oxytoca M5A1 containingchromosomally integrated genes for ethanol production from Zymomonasmobilis (pdc, adhB) and endoglucanase genes from Erwinia chrysanthemi(celY, celZ) is demonstrated to produced over 20,000 U/L ofendoglucanase activity during fermentation. In particular, this strainis demonstrated to, in combination with its native ability to metabolizecellobiose and cellotriose, ferment amorphous cellulose to ethanol(58-76% of theoretical yield) without the addition of cellulase enzymesfrom other-organisms.

Throughout this example, the following materials and methods are usedunless otherwise stated.

Materials and Methods

Bacteria, Plasmids and Culture Conditions

Four ethanologenic derivatives of Klebsiella oxytoca M5A1 were used inthis study. Strain P2 contains chromosomally integrated pdc and adhBgenes from Z. mobilis for ethanol production (Wood et al., (1992) Appl.Environ. Microbiol. 58: 2103-2110). This strain was the parent organismfor three strains that contain highly expressed, chromosomallyintegrated endoglucanase genes from E. chrysanthemi (Zhou et al. (2001)Appl. Environ. Microbiol. 67: 6-14). Strains SZ6, SZ22, and SZ21 containcelZ alone, celY alone, and both celY and celZ, respectively. Additionalgenes (approximately 15 out genes) were required for the efficientsecretion of endoglucanase CelZ (He et al. (1991) Proc. Natl. Acad. Sci.USA 88:1079-1083; Pugsley et al., (1997) Gene 192: 13-19).and weresupplied by plasmid pCPP2006. For consistency, this plasmid has beeninserted into all strains. Recombinant K. oxytoca strains were grown inLuria broth (per liter: 10 g yeast extract, 5 g tryptone, 5 g NaCl)containing a carbohydrate. Spectinomycin (100 mg/L) was included tomaintain plasmid pCPP2006. Seed cultures (5% glucose, 12-16 h) andfermentations were incubated at 35° C. (120 rpm) and were vented using a22 gauge needle.

Assay of Endoglucanase Activity

Total endoglucanase activity (Cel Y and CelZ) was determined forcultures grown for 24 hours in Luria broth containing 5% sorbitol tominimize interference during the determination of reducing sugars (Zhouet al. (2000) J. Bacteriol. 182: 5676-5682). Cell suspensions in brothwere briefly treated with ultrasound to release bound enzymes. Dilutionswere assayed at 35° C. in 50 mM sodium citrate buffer (pH 5.2) usingcarboxymethyl cellulose as the substrate (2%). Reactions were terminatedby heating (10 min, 100° C.). Activity was expressed as gmole per min ofreducing sugar (U) using glucose as a standard.

Thin Layer Chromatography (TLC) Analysis of Cellobiosides

Cellobiosides were separated using Whatman LK5 silica gel plates (Cat.No. 4855-620). Plates were developed in solvent (6 parts chloroform, 7parts acetic acid, and 1 part water), air-dried, and developed a secondtime in the same solvent. Saccharides were visualized by spraying with6.5 mM N-(1-naphthyl)ethylenediamine dihydrochloride and heating to 100°C. for 10 min (Zhou et al. supra).

Preparation of Cellobiosides from Microcrystalline Cellulose

Cellulose was converted to cellobiosides based on the method of Pereiraet al. (1988). Sigmacell type 50 cellulose (100 g; Cat. No. S-5504) wasslowly added to 400 g of 72% H₂SO₄ (w/w) and allowed to hydrolyze at 22°C. with stirring for 18 hr. The digested slurry was precipitated byslowly adding to 2.5 L of cold ethanol (100%), centrifuged, and thepellet washed twice with 1 L of cold ethanol. The pellet was dissolvedin 100 ml of H₂O, adjusted to pH 6, and centrifuged at 5000×g for 20 minto remove insoluble fibers. The supernatant containing cellobiosides wasdried and analyzed.

HPLC Analysis of Cellobiosides

Cellobiosides were analyze using a Waters HPLC equipped with arefractive index monitor and digital integrator. Separations wereperformed at 65° C. using a BioRad HPX-42A column with distilled wateras the mobile phase (0.6 ml/min).

Fermentation of Cellobiosides

Initial fermentations used cellobiosides purchased from the SigmaChemical Company (Cat. No. C-8071). Seed cultures (112.5 μL) weretypically combined with 37.5 μL of 4% cellobiosides in a 1.5-mlmicrocentrifuge tube and incubated for 36 h (35° C. and 120 rpm).Samples (50 μL each; 0 h, 10 h, and 36 h) were heated (100° C., 10 min)to inactivate enzymes and centrifuged (10,000×g, 5 min) to remove celldebris. Supernatants (4 μL) were then spotted on TLC plates foranalysis.

Small fermentations (50-ml flasks, 5 ml broth volume) were conducted ina similar fashion with laboratory samples of cellobiosides prepared bysulfuric acid hydrolysis. Cellobiosides (6 g/L total for saccharidesless than 7 glucosyl residues) were filter sterilized in Luria broth.Flasks were inoculated with pelleted cells (5,000×g, 10 min) from a seedculture (dry weight of 0.33 g/L) to eliminate ethanol produced from theglucose in seed media. Samples were removed initially, and then at 24-hintervals for the determination of the presence of ethanol by gaschromatography (Beall et al. (1991) Biotechnol Bioeng. 38: 296-303).

Preparation of Amorphous Cellulose

Phosphoric acid-swollen cellulose was prepared by a modification of themethod described by Wood (1988). Approximately 20 g of Avicel (Cat. No.PH105, Fluka Chemika) was added slowly to 500 ml 85% H₃PO₄, stirred atroom temperature overnight, poured into 4 L of ice cold water, andallowed to settle without agitation for 30 min. After decanting theupper liquid layer, 4 L of cold water was added and mixed thoroughly,and repeated 5 times with water, once with 1% NaHCO₃ , and 5 more timeswith water (final pH 6-7). The cellulose suspension was concentrated bycentrifugation (5000×g, 20 min) and used as a substrate forfermentation. This viscous suspension contained a mixture of crystallineand amorphous cellulose.

The fraction of amorphous cellulose present in the phosphoricacid-swollen cellulose was estimated by repeated digestion with CelY andCelZ endoglucanases. A 24-h culture of SZ21(pCPP2006) was sonicatedbriefly, centrifuged (5,000×g, 10 min), and the supernatant used as asource of endoglucanase (˜20 U/ml). Approximately 1 g of the viscous,acid-swollen cellulose was weighed into each of 6 pre-weighed centrifugetubes. Two served as controls (no enzyme) and were dried to determineinitial dry weight and moisture content. Endoglucanase (9 ml) was addedto 4 tubes and these were incubated at 35° C. for 12 h. Chloroform (2drops) was added to each tube to retard microbial growth. Aftercentrifugation (5,000×g, 20 min), this process was repeated with newenzyme preparations for a total of 6 successive treatments over a 72 hperiod. Tubes were removed at various times, centrifuged, washed oncewith distilled water to remove soluble products and salts, and dried toa constant weight at 70° C. Amorphous cellulose was calculated as thereduction in dry matter resulting from endoglucanase digestion.

Fermentation of Amorphous Cellulose

Acid swollen cellulose (40 g, unsterilized) was combined with 5 ml of a10× concentrate of Luria broth and 5 ml of seed culture (cell dry weightof 0.33 mg/L) in a 125-ml flask. Both spectinomycin (100 μg/ml) andchloramphenicol (40 μg/ml) were added to eliminate contamination. Theviscous mixture was initially mixed using an applicator stick, thenincubated at 35° C. Due to the high initial viscosity, flasks were mixedat 200 rpm during the first 2 h and subsequently, at 120 rpm. Ethanolwas measured by gas chromatography (Beall et al. (1991) Biotechnol.Bioeng. 38:296-303).

Viscosity

The viscosity of acid-swollen cellulose preparations was estimated at22° C. by timing the flow through a vertical 10-ml glass pipette.Solutions of glycerol were used as standards. Flow times of 2 sec to 75sec were observed for fermentation broths, corresponding to viscositiesof 1 to 1,300 centipoise, respectively.

Results

Comparison of Endoglucanase Activities Produced by EthanologenicDerivatives of K. oxytoca

Ethanologenic strains containing endoglucanase genes from E.chrysanthemi produced substantial levels of activity during glucosefermentation. The highest activity was produced by strain SZ21(pCPP2006)containing both celZ and celY, 29.3≅1.6 U/ml of culture. StrainSZ6(pCPP2006) containing celZ alone produced 22.5±1.7 U/ml and strainSZ22(pCPP2006) containing celY ( celZ deletion of strain SZ21) produced1.0±0.1 U/ml. Approximately 60% of the activity was secreted by eachstrain, the balance being cell associated and readily released by mildsonication. No endoglucanase activity was detected in the parent, strainP2(pCPP2006).

Analysis of Cellobiosides

The presence of cellobiosides was analyzed by thin layer chromatographyand by HPLC. Preparations from the Sigma Chemical Company and thoseprepared in our laboratory were similar. Lane 1 of both thin layerchromatograms (FIG. 17, panels A and B ) shows a representativeseparation of the Sigma product at the beginning of fermentation. Smallamounts of glucose and cellobiose were present with intermediate amountsof cellotriose and cellohexaose, and larger amounts of cellotetraose andcellopentaose. The most intense region, however, remained at the origin.This region contains cellobiosides of greater than 6 residues and otheruncharacterized compounds.

HPLC analyses were in agreement with thin layer chromatograms. The Sigmaproduct contained 3% glucose, 3% cellobiose, 13% cellotriose, 25%cellotetraose, 22% cellopentaose, and 9% cellohexaose. Based on peakareas, approximately 25% of the saccharides were longer than 6 residues.Cellobiosides prepared in our laboratory contained 7% glucose, 6%cellobiose, 10% cellotriose, 15% cellotetraose, 12% cellopentaose, 19%cellohexaose, and 30% saccharides longer than 6 glucosyl residues.

Fermentation of Cellobiosides

FIG. 17 shows a thin layer chromatogram comparing the fermentation ofcellobiosides (Sigma Chemical Company) by strain P2(pCPP2006) and bySZ21(pCPP2006), a derivative which secretes both endoglucanases CelY andCelZ. Utilization of glucose, cellobiose, and cellotriose by strain P2was confirmed and is particularly evident after 36 h. However, thisstrain was unable to metabolize longer cellobiosides. In contrast,strain SZ21 degraded and metabolized virtually all of the separatedcellobiosides and a portion of the material at the origin. It isrelevant to note that after 10 h, the levels of cellobiose andcellotriose in the SZ21 fermentation were several-fold higher thaninitially present. This increase was attributed to the hydrolysis oflonger cellobiosides by secreted CelY and CelZ.

The effectiveness of secreted endoglucanases was also examined duringethanol production from laboratory cellobiosides (Table 17). Althoughonly low levels of ethanol were produced due to the low substrateconcentrations, the benefit of CelZ endoglucanase is clearly evident.The parent P2(pCPP2006) and strain SZ22(pCPP2006; CelY only) producedhalf as much ethanol as the two strains that secreted CelZ:SZ6(pCPP2006; CelZ only) and SZ21 (pCPP2006; CelY and CelZ).Endoglucanase CelY was of no benefit using cellobiosides as substrates,consistent with the requirement for a long-chain substrate.

Ethanol yields were estimated based on cellobiosides containing lessthan 7 glucosyl residues (Table 17). An average of 62% of thetheoretical yield was observed for the two strains producingendoglucanase CelZ. Higher yields (over 90%) were previously measuredduring the fermentation of 100 g/L cellobiose by strain P2 (Wood &Ingram 1992). It is likely that the lower yields observed with lowconcentrations of cellobiosides result in part from evaporative lossesand the diversion of a larger fraction of carbon to cell growth.

Amorphous Cellulose Content of Phosphoric Acid Swollen Cellulose

Acid swollen cellulose partially recrystallizes during the removal ofacid and storage due to extensive hydrogen bonding between celluloseribbons. E. chrysanthemi Cel Y and Cel Z hydrolyze amorphous celluloseand carboxymethyl cellulose but are unable to hydrolyze crystallinecellulose. This resistance of crystalline cellulose to endoglucanasehydrolysis was used to estimate the fraction of washed, acid-swollencellulose which remained amorphous as the loss of insoluble material(dry weight). Samples of amorphous cellulose were repeatedly incubatedwith fresh endoglucanase preparations (˜20 U/ml). After an initial 12-htreatment, 28% of the dry weight was solubilized. After four successivetreatments, 43% was solubilized and remained constant for the two finaltreatments. This value, 43%, represents an estimate of the fraction ofcellulose that remained in the amorphous state. The actual fraction ofamorphous cellulose may be somewhat lower since estimates also includelosses of crystalline cellulose which may have occurred duringcentrifugation and washing.

Fermentation of Amorphous Cellulose

The functional expression of high levels of endoglucanase is sufficientto permit the direct conversion of amorphous cellulose to ethanol by twoderivatives of K. oxytoca P2 (Table 17; FIG. 18). Three concentrationsof amorphous cellulose were tested with similar results.

The highest levels were produced by strain SZ21(pCPP2006) which secretesa combination of CelY and CelZ endoglucanases. Lower levels of ethanolwere produced by strain SZ6(pCPP2006) which secretes CeIZ alone. A smallamount of ethanol was produced from residual glucose in the seed mediaby the parent strain, P2(pCPP2006; no endoglucanase) and by strainSZ22(pCPP2006) which secreted only CelY. The combined effects of CelYand CelZ appears to be synergistic for ethanol production and ethanolyield despite the lack of efficacy of CelY alone. Ethanol yields forstrain SZ21 (pCPP2006; both enzymes) ranged from 58%-76% of thetheoretical maximum. This synergistic effect on ethanol production mayresult from increased hydrolysis due to differences in substratespecificity.

Viscosity Changes During the Fermentation of Amorphous Cellulose

Pronounced viscosity changes were observed during the fermentation ofamorphous cellulose by endoglucanase-secreting strains (FIG. 18). Thelargest reduction in viscosity was evident during the first 2 h ofincubation for SZ6(pCPP2006; CelZ alone) and SZ21(pCPP2006; CelY andCelZ), from a viscosity near that of pure glycerol to that near water.Less pronounced changes were observed with SZ22(pCPP2006; CelY alone)and no change was evident with the parent strain P2(pCPP2006) whichserved as a control. Changes in viscosity were essentially completeafter 12 h.

Differences in viscosity were quantified at the end of fermentation forExperiment 4 in Table 17 (FIG. 18, panel C). These varied by threeorders of magnitude. The highest final viscosity was observed forP2(pCPP2006) which does not produce endoglucanase, 1,300 centipoise.Viscosity was reduced by half (500 centipoise) during fermentation withSZ22(pCPP2006) as a result of hydrolysis by endoglucanase CelY alone. Atthe end of fermentation, the viscosities of broths from both strainsthat secrete CelZ were essentially equivalent to that of water, 1centopoise for SZ21(pCPP2006; CelY and CelZ) and 2 centipoise forSZ6(pCPP2006; CelZ alone). Clearly, CelZ alone was more effective thanCelY alone. No further benefit was attributed to the production of CelYin combination with CelZ when compared at the end of fermentation (96 h)although both enzymes function synergistically in the production ofethanol (Table 17; FIG. 18) and during saccharification.

In summary, these results using, e.g., K. oxytoca strain SZ21,demonstrate an advancement toward the goal of producing sufficientcellulase enzymes for the direct bioconversion of cellobiosides andamorphous cellulose to ethanol without the addition of supplementalenzymes. Endoglucanase levels produced by this strain are over 10-foldover those previously reported for engineered strains of yeast and otherbacteria during ethanol fermentation (Brestic-Goachet et al. 1989, Choet al. 1999, Cho & Yoo 1999, Misawa et al. 1988, Su et al. 1993, VanRensburg et al. 1996, 1998). Moreover, the level of endoglucanaseproduced by strain SZ21 is roughly equivalent to 1% of the endoglucanaseactivity present in commercial cellulase concentrates (Nieves et al.1998, Tomme et al. 1995, Wilson et al. 1997).

The effectiveness of strain SZ21 in the hydrolysis of amorphouscellulose results from the combination of two endoglucanase enzymes E.chrysanthemi and function synergistically (Guiseppi et al. 1991, Zhou &Ingram 2000). Moreover, these secreted enzymes function with thephosphotransferase system in K. oxytoca which provides efficient uptakeand metabolism of cellobiose and cellotriose (Wood & Ingram 1992, Lai etal. 1997). TABLE 17 Production of ethanol from cellobiosides and fromamorphous cellulose Substrate Ethanol^(b) Theoretical Expt. StrainSubstrate (g/L) ^(a) N (g/liter) Yield^(c) 1 P2 (pCPP2006)cellobiosides^((d)) 6.0 2  1.06 33 1 SZ6(pCPP2006) cellobiosides 6.0 2 2.04 63 1 SZ21(pCPP2006) cellobiosides 6.0 2  2.02 62 1 SZ22(pCPP2006)cellobiosides 6.0 2  0.93 26 2 P2(pCPP2006) amorphous cellulose 6.85 31.77 ± 0.06 0 2 SZ6(pCPP2006) amorphous cellulose 6.85 3 3.97 ± 0.12**57 2 SZ21(pCPP2006) amorphous cellulose 6.85 3 4.67 ± 0.06** 76 2SZ22(pCPP2006) amorphous cellulose 6.85 3 1.80 ± 0.01 0 3 P2 (pCPP2006)amorphous cellulose 15.34 3 1.85 ± 0.01 0 3 SZ6(pCPP2006) amorphouscellulose 15.34 3 6.07 ± 0.18** 49 3 SZ21(pCPP2006) amorphous cellulose15.34 3 7.84 ± 0.35** 70 3 SZ22(pCPP2006) amorphous cellulose 15.34 31.92 ± 0.02 0 4^(e) P2 (pCPP2006) amorphous cellulose 28.96 1  1.90 0 4SZ6(pCPP2006) amorphous cellulose 28.96 1 10.1** 51 4 SZ21(pCPP2006)amorphous cellulose 28.96 1 11.3** 58 4 SZ22(pCPP2006) amorphouscellulose 28.96 1  1.80 0^(a)Inocula for cellobioside fermentations were harvested bycentrifugation to eliminate seed broth as a source of ethanol. Anaverage of 1.85 g/L ethanol was produced from residual sugar in the seedbroth used to inoculate the three fermentations of amorphous cellulose.^(b)**Indicates that the value is significantly different from that ofthe control strain (P2) lacking genes encoding E. chrysanthemiendoglucanases (p < 0.001).^(c)Yields are calculated as a percentage of theoretical maxima, assumedto be 0.54 g ethanol per gram of cellobiose and 0.56 g ethanol per gramof amorphous cellulose. For amorphous cellulose, yields were computedafter subtracting ethanol produced from sugar in the inocula.^(d)Cellobiosides (g/L) represents the sum of glucose plus cellobiosidescontaining fewer than 7 glucosyl residues.^(e)The highest coefficient of variation in Experiments No. 2 and No. 3was 4.5% of the mean value. By assuming a similar coefficient ofvariation (5% of the measured value), significance was also estimatedfor Experiment No. 3.Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. Moreover, any numberof genetic constructs, host cells, and methods described in U.S. Pat.Nos. 5,821,093; 5,482,846; 5,424,202; 5,028,539; 5,000,000; 5,487,989,5,554,520, and 5,162,516, may be employed in carrying out the presentinvention and are hereby incorporated by reference.

1-65. (canceled)
 66. A method of making a recombinant host cell suitablefor degrading an oligosaccharide comprising: introducing into said hostcell a first heterologous polynucleotide segment encoding a firstendoglucanase having a first degrading activity, wherein said segment isunder the transcriptional control of a surrogate promoter; and a secondheterologous polynucleotide segment comprising a sequence encoding asecond endoglucanase having a second degrading activity, wherein saidsegment is under the transcriptional control of a surrogate promoter,wherein said first and second endoglucanases are expressed such thatsaid first and said second degrading activities are present in a ratiosuch that the degrading of said oligosaccharide by said first and secondendoglucanases is synergized.
 67. The method of claim 65, wherein saidfirst endoglucanase or said second endoglucanase or both said first andsecond endoglucanases are secreted.
 68. The method of claim 66, whereinsaid host cell is ethanologenic.
 69. The method of claim 66, whereinsaid first endoglucanase is encoded by celZ and said secondendoglucanase is encoded by celY, wherein celZ and celY are derived fromErwinia.
 70. The method of claim 66, wherein said surrogate promoter ofsaid first heterologous polynucleotide segment or said secondheterologous polynucleotide segment or both said first and secondpolynucleotide segments, comprises a polynucleotide fragment derivedfrom Zymomonas mobilis.
 71. The method of claim 68, wherein saidrecombinant host cell is suitable for simultaneous saccharification andfermentation.
 72. The method of claim 70 or 71, wherein said host cellis ethanologenic.
 73. A method of making a recombinant host cellintegrant comprising, introducing into said host cell a vectorcomprising the polynucleotide sequence of pLOI2352 (SEQ ID NO: 17); andidentifying a host cell having said vector stably integrated.
 74. Amethod for expressing a endoglucanase in a host cell comprising:introducing into said host cell a vector comprising the polynucleotidesequence of pLOI2306 (SEQ ID NO: 12); and identifying a host cellexpressing said endoglucanase.
 75. A method for producing ethanol froman oligosaccharide source comprising, contacting said oligosaccharidesource with a ethanologenic host cell comprising: a first heterologouspolynucleotide segment encoding a first endoglucanase having a firstdegrading activity, wherein said segment is under the transcriptionalcontrol of a surrogate promoter; and a second heterologouspolynucleotide segment encoding a second endoglucanase having a seconddegrading activity, wherein said segment is under the transcriptionalcontrol of a surrogate promoter, wherein said first and secondendoglucanases are expressed so that said first and said seconddegrading activities are present in a ratio such that the degrading ofsaid oligosaccharide by said first and second endoglucanases issynergized resulting in a degraded oligosaccharide that is fermentedinto ethanol.
 76. The method of claim 75, wherein said firstendoglucanase is encoded by celZ and said second endoglucanase isencoded by celY gene, wherein celZ and celY are derived from Erwinia.77. The method of claim 75, further said host cell further comprising aheterologous polynucleotide segment encoding at least one pul gene orout gene.
 78. The method of claim 75, wherein said host cell is selectedfrom the family Enterobacteriaceae.
 79. The method of claim 75, whereinsaid host cell is Escherichia or Klebsiella.
 80. The method of claim 79,wherein said host cell is selected from the group consisting of E. coliKO4 (ATCC 55123), E. coli KO11 (ATCC 55124), E. coli KO12 (ATCC 55125),and K. oxytoca P2 (ATCC 55307).
 81. The method of claim 75, wherein saidmethod is conducted in an aqueous solution.
 82. The method of claim 75,wherein said oligosaccharide is selected from the group consisting ofcellooligosaccharide, lignocellulose, hemicellulose, cellulose, pectin,and any combination thereof.
 83. The method of claim 75, wherein saidheterologous polynucleotide segment is, or derived from, of pLOI2352(SEQ ID NO: 17).
 84. The method of claim 75, wherein said firstendoglucanase is EGZ and said second endoglucanase is EGY.
 85. Themethod of claim 75 wherein said surrogate promoter of said firstpolynucleotide segment or said second polynucleotide segment, or bothsaid first second polynucleotide segments comprises a polynucleotidefragment derived from Zymomonas mobilis.
 86. A vector comprising thepolynucleotide sequence of a plasmid, or fragment thereof, selected fromgroup consisting of pLOI2311, pLOI1620, pLOI2316, pLOI2317, pLOI2318,pLOI2319, pLOI2320, pLOI2323, pLOI2342, pLOI2348, pLOI2349, pLOI2350,pLOI2352, pLOI2353, pLOI2354, pLOI2355, pLOI2356, pLOI2357, pLO12358,and pLO2359.
 87. A host cell comprising a vector having thepolynucleotide sequence of a plasmid, of fragment thereof, selected fromthe group consisting of pLOI2311, pLOI1620, pLOI2316, pLOI2317,pLOI2318, pLOI2319, pLOI2320, pLOI2323, pLOI2342, pLOI2348, pLOI2349,pLOI2350, pLOI2352, pLOI2353, pLOI2354, pLOI2355, pLOI2356, pLOI2357,pLOI2358, and pLO2359.
 88. The host cell of claim 87, wherein said hostis selected from the group comprising Klebsiella oxytoca strain P2(pCPP2006), Klebsiella oxytoca strain SZ6 (pCPP2006), Klebsiella oxytocastrain SZ21 (pCPP2006), and Klebsiella oxytoca strain SZ22 (pCPP2006).89-110. (canceled)